Interferon-beta variants and conjugates

ABSTRACT

The present invention provides new interferon β conjugates, methods of preparing such conjugates and the use of such conjugates in therapy, in particular for the treatment of multiple sclerosis.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] Pursuant to 35 USC 119 and 120, and any other applicable statuteor rule, this application claims the benefit of and priority from eachof the following Application Numbers/filing dates: Denmark PatentApplication No. PA 1999 01197, filed Aug. 27, 1999; U.S. Ser. No.60/160,782, filed Oct. 21, 1999; Denmark Patent Application No. PA 199901691, filed Nov. 26, 1999; U.S. Ser. No. 60/169,077, filed Dec. 6,1999; Denmark Patent Application No. PA 2000 00194, filed Feb. 7, 2000;Denmark Patent Application No. PA 2000 00363, filed Mar. 7, 2000; U.S.Ser. No. 60/189,599, filed Mar. 15, 2000; Denmark Patent Application No.PA 2000 00642, filed Apr. 14, 2000; and U.S. Ser. No. 60/202,248, filedMay 5, 2000, the disclosures of which are incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] Interferons are important cytokines characterized by antiviral,antiproliferative, and immunomodulatory activities. These activitiesform a basis for the clinical benefits that have been observed in anumber of diseases, including hepatitis, various cancers and multiplesclerosis. The interferons are divided into the type I and type IIclasses. Interferon β belongs to the class of type I interferons, whichalso includes interferons α, τ and ω, whereas interferon γ is the onlyknown member of the distinct type II class.

[0003] Human interferon β is a regulatory polypeptide with a molecularweight of 22 kDa consisting of 166 amino acid residues. It can beproduced by most cells in the body, in particular fibroblasts, inresponse to viral infection or exposure to other biologics. It binds toa multimeric cell surface receptor, and productive receptor bindingresults in a cascade of intracellular events leading to the expressionof interferon β inducible genes which in turn produces effects which canbe classified as antiviral, antiproliferative and immunomodulatory.

[0004] The amino acid sequence of human interferon β was reported byTaniguchi, Gene 10:11-15, 1980, and in EP 83069, EP 41313 and U.S. Pat.No. 4,686,191.

[0005] Crystal structures have been reported for human and murineinterferon β, respectively (Proc. Natl. Acad. Sci. USA 94:11813-11818,1997. J. Mol. Biol. 253:187-207, 1995). They have been reviewed in CellMol. Life Sci. 54:1203-1206, 1998.

[0006] Relatively few protein-engineered variants of interferon β havebeen reported (WO 9525170, WO 9848018, U.S. Pat. Nos. 5,545,723,4,914,033, EP 260350, U.S. Pat. Nos. 4,588,585, 4,769,233, Stewart etal, DNA Vol 6 no2 1987 pp. 119-128, Runkel et al, 1998, Jour. Biol.Chem. 273, No. 14, pp. 8003-8008).

[0007] Expression of interferon β in CHO cells has been reported (U.S.Pat. Nos. 4,966,843, 5,376,567 and 5,795,779).

[0008] Redlich et al, Proc. Natl. Acad. Sci., USA, Vol. 88, pp.4040-4044, 1991 disclose immunoreactivity of antibodies againstsynthetic peptides corresponding to peptide stretches of recombinanthuman interferon β with the mutation C17S.

[0009] Interferon β molecules with a particular glycosylation patternand methods for their preparation have been reported (EP 287075 and EP539300).

[0010] Various references disclose modification of polypeptides bypolymer conjugation or glycosylation. Polymer modification of nativeinterferon β or a C17S variant thereof has been reported (EP 229108,U.S. Pat. No. 5,382,657, EP 593868, U.S. Pat. No. 4,917,888 and WO99/55377). U.S. Pat. No. 4,904,584 discloses PEGylated lysine depletedpolypeptides, wherein at least one lysine residue has been deleted orreplaced with any other amino acid residue. WO 99/67291 discloses aprocess for conjugating a protein with PEG, wherein at least one aminoacid residue on the protein is deleted and the protein is contacted withPEG under conditions sufficient to achieve conjugation to the protein.WO 99/03887 discloses PEGylated variants of polypeptides belonging tothe growth hormone superfamily, wherein a cysteine residue has beensubstituted with a non-essential amino acid residue located in aspecified region of the polypeptide. Interferon β is mentioned as oneexample of a polypeptide belonging to the growth hormone superfamily. WO00/23114 discloses glycosylated and pegylated interferon β. WO 00/23472discloses interferon β fusion proteins. WO 00/26354 discloses a methodof producing a glycosylated polypeptide variant with reducedallergenicity, which as compared to a corresponding parent polypeptidecomprises at least one additional glycosylation site. U.S. Pat. No.5,218,092 discloses modification of granulocyte colony stimulatingfactor (G-CSF) and other polypeptides so as to introduce at least oneadditional carbohydrate chain as compared to the native polypeptide.Interferon β is mentioned as one example among many polypeptides whichallegedly can be modified according to the technology described in U.S.Pat. No. 5,218,092.

[0011] Commercial preparations of interferon β are sold under the namesBetaseron® (also termed interferon β1b, which is non-glycosylated,produced using recombinant bacterial cells, has a deletion of theN-terminal methionine residue and the C17S mutation), and Avonex™ andRebif® (also termed interferon β1a, which is glycosylated, producedusing recombinant mammalian cells) for treatment of patients withmultiple sclerosis, and have been shown to be effective in reducing theexacerbation rate. More patients treated with these interferon β agentsremain exacerbation-free for prolonged periods of time as compared withplacebo-treated patients. Furthermore, the accumulation rate ofdisability is reduced (Neurol. 51:682-689, 1998).

[0012] Comparison of interferon β1a and β1b with respect to structureand function has been presented in Pharmaceut. Res. 15:641-649, 1998.

[0013] Interferon β is the first therapeutic intervention shown to delaythe progression of multiple sclerosis, a relapsing then progressiveinflammatory degenerative disease of the central nervous system.

[0014] Its mechanism of action, however, remains largely unclear. Itappears that interferon β has inhibitory effects on the proliferation ofleukocytes and antigen presentation. Furthermore, interferon β maymodulate the profile of cytokine production towards an anti-inflammatoryphenotype. Finally, interferon β can reduce T-cell migration byinhibiting the activity of T-cell matrix metalloproteases. Theseactivities are likely to act in concert to account for the mechanism ofinterferon β in MS (Neurol. 51:682-689, 1998).

[0015] In addition, interferon β may be used for the treatment ofosteosarcoma, basal cell carcinoma, cervical dysplasia, glioma, acutemyeloid leukemia, multiple myeloma, Hodgkin's disease, breast carcinoma,melanoma, and viral infections such as papilloma virus, viral hepatitis,herpes genitalis, herpes zoster, herpetic keratitis, herpes simplex,viral encephalitis, cytomegalovirus pneumonia, and rhinovirus.

[0016] Various side effects are associated with the use of currentpreparations of interferon β, including injection site reactions, fever,chills, myalgias, arthralgias, and other flu-like symptoms (Clin.Therapeutics, 19:883-893, 1997).

[0017] In addition, 6-40% of patients develop neutralizing antibodies tointerferon β (Int. Arch. Allergy Immunol. 118:368-371, 1999). It hasbeen shown that development of interferon β-neutralizing antibodiesdecreases the biological response to interferon β, and cause a trendtowards decreased treatment efficacy (Neurol. 50:1266-1272, 1998).Neutralizing antibodies will likely also impede the therapeutic utilityof interferon β in connection with treatment of other diseases (Immunol.Immuther. 39:263-268, 1994).

[0018] Given the magnitude of side effects with current interferon βproducts, their association with frequent injection, the risk ofdeveloping neutralizing antibodies impeding the desired therapeuticeffect of interferon β, and the potential for obtaining more optimaltherapeutic interferon β levels with concomitant enhanced therapeuticeffect, there is clearly a need for improved interferon β-likemolecules.

SUMMARY OF THE INVENTION

[0019] This application discloses improved interferon β moleculesproviding one or more of the aforementioned desired benefits. Inparticular conjugates are disclosed that exhibit interferon β activityand comprise at least one non-polypeptide moiety covalently attached toan interferon β polypeptide that comprises an amino acid sequence thatdiffers from that of wildtype human interferon β with the amino acidsequence shown in SEQ ID NO 2 in at least one amino acid residueselected from an introduced or removed amino acid residue comprising anattachment group for the non-polypeptide moiety. Conjugates of thepresent invention have a number of improved properties as compared tohuman interferon β, including reduced immunogenicity, increasedfunctional in vivo half-life, increased serum half-life, and/orincreased bioavailability. Consequently, the conjugate of the inventionoffers a number of advantages over the currently available interferon βcompounds, including longer duration between injections, fewer sideeffects, and/or increased efficiency due to reduction in antibodies.Moreover, higher doses of active protein and thus a more effectivetherapeutic response may be obtained by use of a conjugate of theinvention. Furthermore, conjugates of the invention have demonstratedsignificantly reduced cross-reactivity with sera from patients treatedwith currently available interferon β products as defined hereinbelow.

[0020] In one aspect the invention relates to a conjugate exhibitinginterferon β activity and comprising at least one first non-polypeptidemoiety covalently attached to an interferon β polypeptide, the aminoacid sequence of which differs from that of wild-type human interferon βin at least one introduced and at least one removed amino acid residuecomprising an attachment group for said first non-polypeptide moiety.

[0021] In another aspect the invention relates to a conjugate exhibitinginterferon β activity and comprising at least one first non-polypeptidemoiety conjugated to at least one lysinc residue of an interferon βpolypeptide, the amino acid sequence of which differs from that ofwild-type human interferon β in at least one introduced and/or at leastone removed lysine residue.

[0022] In yet another aspect the invention relates to a conjugateexhibiting interferon β activity and comprising at least one firstnon-polypeptide moiety conjugated to at least one cysteine residue of aninterferon β polypeptide, the amino acid sequence of which differs fromat least one introduce cysteine residue into a position that inwild-type human interferon β is occupied by a surface exposed amino acidresidue.

[0023] In yet another aspect the invention relates to a conjugateexhibiting interferon β activity and comprising at least one firstnon-polypeptide moiety having an acid group as an attachment group,which moiety is conjugated to at least one aspartic acid or glutamicacid residue of an interferon β polypeptide, the amino acid sequence ofwhich differs from that of wild-type human interferon β in at least oneintroduced and/or at least one removed aspartic acid or glutamic acidresidue.

[0024] In yet another aspect the invention relates to a conjugateexhibiting interferon β activity and comprising at least one polymermolecule and at least one sugar moiety covalently attached to aninterferon β polypeptide, the amino acid sequence of which differs fromthat of wild-type human interferon β in

[0025] (a) at least one introduced and/or at least one removed aminoacid residue comprising an attachment group for the polymer molecule,and

[0026] (b) at least one introduced and/or at least one removed aminoacid residue comprising an attachment group for the sugar moiety,

[0027] provided that when the attachment group for the polymer moleculeis a cysteine residue, and the sugar moiety is an N-linked sugar moiety,a cysteine residue is not inserted in such a manner that anN-glycosylation site is destroyed.

[0028] In yet another aspect the invention relates to a conjugateexhibiting interferon β activity and comprising an interferon βpolypeptide, the amino acid sequence of which differs from that ofwild-type human interferon β in at least one introduced glycosylationsite, the conjugate further comprising at least one un-PEGylated sugarmoiety attached to an introduced glycosylation site.

[0029] In yet another aspect the invention relates to a conjugateexhibiting interferon β activity and comprising an interferon βpolypeptide, the amino acid sequence of which differs from that ofwild-type human interferon β in that a glycosylation site has beenintroduced or removed by way of introduction or removal of amino acidresidue(s) constituting a part of a glycosylation site in a positionthat in wildtype human interferon β is occupied by a surface exposedamino acid residue.

[0030] In a still further aspect the invention relates to a conjugateexhibiting interferon β activity and comprising a sugar moietycovalently attached to an interferon β polypeptide, the amino acidsequence of which differs from that of wild-type human interferon β inat least one removed glycosylation site.

[0031] In still further aspects the invention relates to means andmethods for preparing a conjugate or interferon β polypeptide for use inthe invention, including nucleotide sequences and expression vectorsencoding the polypeptide as well as methods for preparing thepolypeptide or the conjugate.

[0032] In final aspects the invention relates to a therapeuticcomposition comprising a conjugate of the invention, to a conjugate orcomposition of the invention for use in therapy, to the use of aconjugate or composition in therapy or for the manufacture of amedicament for treatment of diseases.

BRIEF DESCRIPTION OF THE FIGURES

[0033]FIG. 1 illustrates the antiviral activity of a conjugate of theinvention,

[0034]FIG. 2 depicts the yield of interferon β production obtainedaccording to Example 8.

DETAILED DISCUSSION

[0035] Definitions

[0036] In the context of the present application and invention thefollowing definitions apply:

[0037] The term “conjugate” (or interchangeably “conjugatedpolypeptide”) is intended to indicate a heterogeneous (in the sense ofcomposite or chimeric) molecule formed by the covalent attachment of oneor more polypeptide(s) to one or more non-polypeptide moieties. The termcovalent attachment means that the polypeptide and the non-polypeptidemoiety are either directly covalently joined to one another, or else areindirectly covalently joined to one another through an interveningmoiety or moieties, such as a bridge, spacer, or linkage moiety ormoieties using an attachment group present in the polypeptide.Preferably, the conjugate is soluble at relevant concentrations andconditions, i.e. soluble in physiological fluids such as blood. Examplesof conjugated polypeptides of the invention include glycosylated and/orPEGylated polypeptides. The term “non-conjugated polypeptide” may beused about the polypeptide part of the conjugate.

[0038] The term “non-polypeptide moiety” is intended to indicate amolecule that is capable of conjugating to an attachment group of thepolypeptide of the invention. Preferred examples of such moleculeinclude polymer molecules, sugar moieties, lipophilic compounds, ororganic derivatizing agents. When used in the context of a conjugate ofthe invention it will be understood that the non-polypeptide moiety islinked to the polypeptide part of the conjugate through an attachmentgroup of the polypeptide.

[0039] The term “polymer molecule” is defined as a molecule formed bycovalent linkage of two or more monomers, wherein none of the monomersis an amino acid residue, except where the polymer is human albumin oranother abundant plasma protein. The term “polymer” may be usedinterchangeably with the term “polymer molecule”. The term is intendedto cover carbohydrate molecules attached by in vitro glycosylation, i.e.a synthetic glycosylation performed in vitro normally involvingcovalently linking a carbohydrate molecule to an attachment group of thepolypeptide, optionally using a cross-linking agent. Carbohydratemolecules attached by in vivo glycosylation, such as N- orO-glycosylation (as further described below)) are referred to herein as“a sugar moiety”. Except where the number of non-polypeptide moieties,such as polymer molecule(s) or sugar moieties in the conjugate isexpressly indicated every reference to “a non-polypeptide moiety”contained in a conjugate or otherwise used in the present inventionshall be a reference to one or more non-polypeptide moieties, such aspolymer molecule(s) or sugar moieties, in the conjugate.

[0040] The term “attachment group” is intended to indicate an amino acidresidue group of the polypeptide capable of coupling to the relevantnon-polypeptide moiety. For instance, for polymer, in particular PEG,conjugation a frequently used attachment group is the ε-amino group oflysine or the N-terminal amino group. Other polymer attachment groupsinclude a free carboxylic acid group (e.g. that of the C-terminal aminoacid residue or of an aspartic acid or glutamic acid residue), suitablyactivated carbonyl groups, oxidized carbohydrate moieties and mercaptogroups.

[0041] For in vivo N-glycosylation, the term “attachment group” is usedin an unconventional way to indicate the amino acid residuesconstituting an N-glycosylation site (with the sequence N—X′—S/T/C—X″,wherein X′ is any amino acid residue except proline, X″ any amino acidresidue that may or may not be identical to X′ and preferably isdifferent from proline, N is asparagine and S/T/C is either serine,threonine or cysteine, preferably serine or threonine, and mostpreferably threonine). Although the asparagine residue of theN-glycosylation site is the one to which the sugar moiety is attachedduring glycosylation, such attachment cannot be achieved unless theother amino acid residues of the N-glycosylation site is present.Accordingly, when the non-polypeptide moiety is an N-linked sugarmoiety, the term “amino acid residue comprising an attachment group forthe non-polypeptide moiety” as used in connection with alterations ofthe amino acid sequence of the parent polypeptide is to be understood asamino acid residues constituting an N-glycosylation site is/are to bealtered in such a manner that either a functional N-glycosylation siteis introduced into the amino acid sequence or removed from saidsequence. For an “O-glycosylation site” the attachment group is theOH-group of a serine or threonine residue.

[0042] The term “one difference” or “differs from” as used in connectionwith specific mutations is intended to allow for additional differencesbeing present apart from the specified amino acid difference. Forinstance, in addition to the removal and/or introduction of amino acidresidues comprising an attachment group for the non-polypeptide moietythe interferon β polypeptide may comprise other substitutions that arenot related to introduction and/or removal of such amino acid residues.The term “at least one” as used about a non-polypeptide moiety, an aminoacid residue, a substitution, etc is intended to mean one or more. Theterms “mutation” and “substitution” are used interchangeably herein.

[0043] In the present application, amino acid names and atom names (e.g.CA, CB, CD, CG, SG, NZ, N, O, C, etc) are used as defined by the ProteinDataBank (PDB) (www.pdb.org) which are based on the IUPAC nomenclature(IUPAC Nomenclature and Symbolism for Amino Acids and Peptides (residuenames, atom names e.t.c.), Eur. J. Biochem., 138, 9-37 (1984) togetherwith their corrections in Eur. J. Biochem., 152, 1 (1985). CA issometimes referred to as Cα, CB as Cβ. The term “amino acid residue” isintended to indicate an amino acid residue contained in the groupconsisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid(Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine(Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys orK), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N),proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine(Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp orW), and tyrosine (Tyr or Y) residues. The terminology used foridentifying amino acid positions/substitutions is illustrated asfollows: C17 (indicates position #17 occupied by a cysteine residue inthe amino acid sequence shown in SEQ ID NO 2). C17S (indicates that thecysteine residue of position 17 has been replaced with a serine). Thenumbering of amino acid residues made herein is made relative to theamino acid sequence shown in SEQ ID NO 2. “M1del” is used about adeletion of the methionine residue occupying position 1. Multiplesubstitutions are indicated with a “+”, e.g. R71N+D73T/S means an aminoacid sequence which comprises a substitution of the arginine residue inposition 71 with an asparagine and a substitution of the aspartic acidresidue in position 73 with a threonine or serine residue, preferably athreonine residue. T/S as used about a given substitution herein meanseither a T or a S residue, preferably a T residue.

[0044] The term “nucleotide sequence” is intended to indicate aconsecutive stretch of two or more nucleotide molecules. The nucleotidesequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin,or any combinations thereof.

[0045] The term “interferon β protein sequence family” is used in itsconventional meaning, i.e. to indicate a group of polypeptides withsufficiently homologous amino acid sequences to allow alignment of thesequences, e.g. using the CLUSTALW program. An interferon β sequencefamily is available, e.g. from the PFAM families, version 4.0, or may beprepared by use of a suitable computer program such as CLUSTALW version1.74 using default parameters (Thompson et al., 1994, CLUSTAL W:improving the sensitivity of progressive multiple sequence alignmentthrough sequence weighting, position-specific gap penalties and weightmatrix choice, Nucleic Acids Research, 22:4673-4680).

[0046] The term “polymerase chain reaction” or “PCR” generally refers toa method for amplification of a desired nucleotide sequence in vitro, asdescribed, for example, in U.S. Pat. No. 4,683,195. In general, the PCRmethod involves repeated cycles of primer extension synthesis, usingoligonucleotide primers capable of hybridising preferentially to atemplate nucleic acid.

[0047] “Cell”, “host cell”, “cell line” and “cell culture” are usedinterchangeably herein and all such terms should be understood toinclude progeny resulting from growth or culturing of a cell.“Transformation” and “transfection” are used interchangeably to refer tothe process of introducing DNA into a cell.

[0048] “Operably linked” refers to the covalent joining of two or morenucleotide sequences, by means of enzymatic ligation or otherwise, in aconfiguration relative to one another such that the normal function ofthe sequences can be performed. For example, the nucleotide sequenceencoding a presequence or secretory leader is operably linked to anucleotide sequence for a polypeptide if it is expressed as a preproteinthat participates in the secretion of the polypeptide: a promoter orenhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the nucleotidesequences being linked are contiguous and, in the case of a secretoryleader, contiguous and in reading phase. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,then synthetic oligonucleotide adaptors or linkers are used, inconjunction with standard recombinant DNA methods.

[0049] The term “introduce” is primarily intended to mean substitutionof an existing amino acid residue, but may also mean insertion of anadditional amino acid residue. The term “remove” is primarily intendedto mean substitution of the amino acid residue to be removed by anotheramino acid residue, but may also mean deletion (without substitution) ofthe amino acid residue to be removed.

[0050] The term “immunogenicity” as used in connection with a givensubstance is intended to indicate the ability of the substance to inducea response from the immune system. The immune response may be a cell orantibody mediated response (see, e.g., Roitt: Essential Immunology(8^(th) Edition, Blackwell) for further definition of immunogenicity).Immunogenicity may be determined by use of any suitable method known inthe art, e.g. in vivo or in vitro, e.g. using the in vitroimmunogenicity test outlined in the Materials and Methods section below.The term “reduced immunogenicity” is intended to indicate that theconjugate or polypeptide of the present invention gives rise to ameasurably lower immune response than a reference molecule, such aswildtype human interferon β, e.g. Rebif or Avonex, or a variant ofwild-type human interferon β such as Betaseron, as determined undercomparable conditions. When reference is made herein to commerciallyavailable interferon β products (i.e. Betaseron, Avonex and Rebif), itshould be understood to mean either the formulated product or theinterferon β polypeptide part of the product (as appropriate). Normally,reduced antibody reactivity (e.g. reactivity towards antibodies presentin serum from patients treated with commercial interferon β products) isan indication of reduced immunogenicity.

[0051] The term “functional in vivo half-life” is used in its normalmeaning, i.e. the time at which 50% of a given functionality of thepolypeptide or conjugate is retained (such as the time at which 50% ofthe biological activity of the polypeptide or conjugate is still presentin the body/target organ, or the time at which the activity of thepolypeptide or conjugate is 50% of the initial value). As an alternativeto determining functional in vivo half-life, “serum half-life” may bedetermined, i.e. the time in which 50% of the polypeptide or conjugatemolecules circulate in the plasma or bloodstream prior to being cleared.Determination of serum half-life is often more simple than determiningfunctional in vivo half-life and the magnitude of serum half-life isusually a good indication of the magnitude of functional in vivohalf-life. Alternative terms to serum half-life include “plasmahalf-life”, “circulating half-life”, “serum clearance”, “plasmaclearance” and “clearance half-life”. The functionality to be retainedis normally selected from antiviral, antiproliferative, immunomodulatoryor receptor binding activity. Functional in vivo half-life and serumhalf-life may be determined by any suitable method known in the art asfurther discussed in the Materials and Methods section hereinafter.

[0052] The polypeptide or conjugate is normally cleared by the action ofone or more of the reticuloendothelial systems (RES), kidney, spleen orliver, or by specific or unspecific proteolysis. Clearance taking placeby the kidneys may also be referred to as “renal clearance” and is e.g.accomplished by glomerular filtration, tubular excretion or tubularelimination. Normally, clearance depends on physical characteristics ofthe conjugate, including molecular weight, size (diameter) (relative tothe cut-off for glomerular filtration), charge, symmetry,shape/rigidity, attached carbohydrate chains, and the presence ofcellular receptors for the protein. A molecular weight of about 67 kDais considered to be an important cut-off-value for renal clearance.

[0053] Reduced renal clearance may be established by any suitable assay,e.g. an established in vivo assay. Typically, the renal clearance isdetermined by administering a labelled (e.g. radiolabelled orfluorescence labelled) polypeptide conjugate to a patient and measuringthe label activity in urine collected from the patient. Reduced renalclearance is determined relative to the corresponding non-conjugatedpolypeptide or the non-conjugated corresponding wild-type polypeptide ora commercial interferon β product under comparable conditions.

[0054] The term “increased” when used with respect to the functional invivo half-life or serum half-life is used to indicate that the relevanthalf-life of the conjugate or polypeptide is statistically significantlyincreased relative to that of a reference molecule, such as anunconjugated wildtype human interferon β (e.g. Avonex™ or Rebif®) or anunconjugated variant human interferon β (e.g. Betaseron®) as determinedunder comparable conditions.

[0055] The term “reduced immunogenicity and/or increased functional invivo half-life and/or increased serum half-life” is to be understood ascovering any one, two or all of these properties. Preferably, aconjugate or polypeptide of the invention has at least two or theseproperties, i.e. reduced immunogenicity and increased functional in vivohalf-life, reduced immunogenicity and increased serum half-life orincreased functional in vivo half-life and increased serum half-life.Most preferably, the conjugate or polypeptide of the invention has allproperties.

[0056] The term “exhibiting interferon β activity” is intended toindicate that the polypeptide or conjugate has one or more of thefunctions of native interferon β, in particular human wildtypeinterferon β with the amino acid sequence shown in SEQ ID NO 2 (which isthe mature sequence) optionally expressed in a glycosylating host cellor any of the commercially available interferon β products. Suchfunctions include capability to bind to an interferon receptor that iscapable of binding interferon β and initiating intracellular signalingfrom the receptor, in particular a type I interferon receptorconstituted by the receptor subunits IFNAR-2 and IFNAR-1 (Domanski etal., The Journal of Biological Chemistry, Vol. 273, No. 6, pp3144-3147,1998, Mogensen et al., Journal of Interferon and Cytokine Research, 19:1069-1098, 1999), and antiviral, antiproliferative or immunomodulatoryactivity (which can be determined using assays known in the art (e.g.those cited in the following disclosure)). Interferon β activity may beassayed by methods known in the art as exemplified in the Materials andMethods section hereinafter.

[0057] The polypeptide or conjugate “exhibiting” or “having” interferonβ activity is considered to have such activity, when it displays ameasurable function, e.g. a measurable receptor binding and stimulatingactivity (e.g. as determined by the primary or secondary assay describedin the Materials and Methods section). The polypeptide exhibitinginterferon β activity may also be termed “interferon β molecule” or“interferon β polypeptide” herein. The term “interferon β polypeptide”is primarily used herein about modified polypeptides of the invention(having introduced or removed attachment groups for the relevantnon-polypeptide moiety).

[0058] The term “parent interferon β” is intended to indicate thestarting molecule to be improved in accordance with the presentinvention. While the parent interferon β may be of any origin, such asvertebrate or mammalian origin (e.g. any of the origins defined in WO00/23472), the parent interferon β is preferably wild-type humaninterferon β with SEQ ID NO 2 or a variant thereof. A “variant” is apolypeptide, which differs in one or more amino acid residues from aparent polypeptide, normally in 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 amino acid residues. Examples of wild-type human interferonβ include the polypeptide part of Avonex™ or Rebif®. An example of aparent interferon β variant is Betaseron®. Alternatively, the parentinterferon β polypeptide may comprise an amino acid sequence, which is ahybrid molecule between interferon β and another homologous polypeptide,such as interferon α, optionally containing one or more additionalsubstitutions introduced into the hybrid molecule. Such a hybridmolecule may contain an amino acid sequence, which differs in more than10 amino acid residues from the amino acid sequence shown in SEQ ID NO2. In order to be useful in the present invention the hybrid moleculeexhibits interferon β activity (e.g. as determined in the secondaryassay described in the Materials and Methods section herein).

[0059] The term “functional site” as used about a polypeptide orconjugate of the invention is intended to indicate one or more aminoacid residues which is/are essential for or otherwise involved in thefunction or performance of interferon β, and thus “located at” thefunctional site. The functional site is e.g. a receptor binding site andmay be determined by methods known in the art, preferably by analysis ofa structure of the polypeptide complexed to a relevant receptor, such asthe type I interferon receptor constituted by IFNAR-1 and IFNAR-2.

CONJUGATE OF THE INVENTION

[0060] As stated above, in a first aspect, the invention relates to aconjugate exhibiting interferon β activity and comprising at least onefirst non-polypeptide moiety covalently attached to an interferon βpolypeptide, the amino acid sequence of which differs from that ofwildtype human interferon β in at least one introduced and at least oneremoved amino acid residue comprising an attachment group for said firstnon-polypeptide moiety.

[0061] By removing and/or introducing amino acid residues comprising anattachment group for the non-polypeptide moiety it is possible tospecifically adapt the polypeptide so as to make the molecule moresusceptible to conjugation to the non-polypeptide moiety of choice, tooptimize the conjugation pattern (e.g. to ensure an optimal distributionof non-polypeptide moieties on the surface of the interferon β moleculeand thereby, e.g., effectively shield epitopes and other surface partsof the polypeptide without significantly impairing the functionthereof). For instance, by introduction of attachment groups, theinterferon β polypeptide is boosted or otherwise altered in the contentof the specific amino acid residues to which the relevantnon-polypeptide moiety binds, whereby a more efficient, specific and/orextensive conjugation is achieved. By removal of one or more attachmentgroups it is possible to avoid conjugation to the non-polypeptide moietyin parts of the polypeptide in which such conjugation isdisadvantageous, e.g. to an amino acid residue located at or near afunctional site of the polypeptide (since conjugation at such a site mayresult in inactivation or reduced interferon β activity of the resultingconjugate due to impaired receptor recognition). Further, it may beadvantageous to remove an attachment group located closely to anotherattachment group in order to avoid heterogeneous conjugation to suchgroups.

[0062] It will be understood that the amino acid residue comprising anattachment group for a non-polypeptide moiety, either it be removed orintroduced, is selected on the basis of the nature of thenon-polypeptide moiety and, in most instances, on the basis of theconjugation method to be used. For instance, when the non-polypeptidemoiety is a polymer molecule, such as a polyethylene glycol orpolyalkylene oxide derived molecule, amino acid residues capable offunctioning as an attachment group may be selected from the groupconsisting of lysine, cysteine, aspartic acid, glutamic acid andarginine. When the non-polypeptide moiety is a sugar moiety theattachment group is an in vivo glycosylation site, preferably anN-glycosylation site.

[0063] Whenever an attachment group for a non-polypeptide moiety is tobe introduced into or removed from the interferon β polypeptide inaccordance with the present invention, the position of the interferon βpolypeptide to be modified is conveniently selected as follows:

[0064] The position is preferably located at the surface of theinterferon β polypeptide and more preferably occupied by an amino acidresidue which has more than 25% of its side chain exposed to thesolvent, preferably more than 50% of its side chain exposed to thesolvent. Such positions have been identified on the basis of an analysisof a 3D structure of the human interferon β molecule as described in theMethods section herein.

[0065] Alternatively or additionally, the position to be modified isidentified on the basis of an analysis of an interferon β proteinsequence family. More specifically, the position to be modified can beone, which in one or more members of the family other than the parentinterferon β, is occupied by an amino acid residue comprising therelevant attachment group (when such amino acid residue is to beintroduced) or which in the parent interferon β, but not in one or moreother members of the family, is occupied by an amino acid residuecomprising the relevant attachment group (when such amino acid residueis to be removed).

[0066] In order to determine an optimal distribution of attachmentgroups, the distance between amino acid residues located at the surfaceof the interferon β molecule is calculated on the basis of a 3Dstructure of the interferon β polypeptide. More specifically, thedistance between the CB's of the amino acid residues comprising suchattachment groups, or the distance between the functional group (NZ forlysine, CG for aspartic acid, CD for glutamic acid, SG for cysteine) ofone and the CB of another amino acid residue comprising an attachmentgroup are determined. In case of glycine, CA is used instead of CB. Inthe interferon β polypeptide part of a conjugate of the invention, anyof said distances is preferably more than 8 Å, in particular more than10 Å in order to avoid or reduce heterogeneous conjugation.

[0067] Furthermore, in the interferon β polypeptide part of a conjugateof the invention attachment groups located at the receptor-binding siteof interferon β has preferably been removed, preferably by substitutionof the amino acid residue comprising such group.

[0068] A still further generally applicable approach for modifying aninterferon β polypeptide is to shield, and thereby destroy or otherwiseinactivate an epitope present in the parent interferon β, by conjugationto a non-polypeptide moiety. Epitopes of human interferon β may beidentified by use of methods known in the art, also known as epitopemapping, see, e.g. Romagnoli et al., J. Biol Chem, 1999, 380(5):553-9,DeLisser HM, Methods Mol Biol, 1999, 96:11-20, Van de Water et al., ClinImmunol Immunopathol, 1997, 85(3):229-35, Saint-Remy JM, Toxicology,1997, 119(l):77-81, and Lane D P and Stephen C W, Curr Opin Immunol,1993, 5(2):268-71. One method is to establish a phage display libraryexpressing random oligopeptides of e.g. 9 amino acid residues. IgG1antibodies from specific antisera towards human interferon β arepurified by immunoprecipitation and the reactive phages are identifiedby immunoblotting. By sequencing the DNA of the purified reactivephages, the sequence of the oligopeptide can be determined followed bylocalization of the sequence on the 3D-structure of the interferon β.Alternatively, epitopes can be identified according to the methoddescribed in U.S. Pat. No. 5,041,376. The thereby identified region onthe structure constitutes an epitope that then can be selected as atarget region for introduction of an attachment group for thenon-polypeptide moiety. Preferably, at least one epitope, such as two,three or four epitopes of human recombinant interferon β (optionallycomprising the C17S mutation) are shielded by a non-polypeptide moietyaccording to the present invention. Accordingly, in one embodiment, theconjugate of the invention has at least one shielded epitope as comparedto wild type human interferon β, optionally comprising the C17Smutation, including any commercially available interferon β. Preferably,the conjugate of the invention comprises a polypeptide that is modifiedso as to shield the epitope located in the vicinity of amino acidresidue Q49 and/or F111. This may be done by introduction of anattachment group for a non-polypeptide moiety into a position located inthe vicinity of (i.e. within 4 amino acid residues in the primarysequence or within about 10 Å in the tertiary sequence) of Q49 and/orF111. The 10 Å distance is measured between CB's (CA's in case ofglycine). Such specific introductions are described in the followingsections.

[0069] In case of removal of an attachment group, the relevant aminoacid residue comprising such group and occupying a position as definedabove is preferably substituted with a different amino acid residue thatdoes not comprise an attachment group for the non-polypeptide moiety inquestion.

[0070] In case of introduction of an attachment group, an amino acidresidue comprising such group is introduced into the position,preferably by substitution of the amino acid residue occupying suchposition.

[0071] The exact number of attachment groups available for conjugationand present in the interferon β polypeptide is dependent on the effectdesired to be achieved by conjugation. The effect to be obtained is,e.g., dependent on the nature and degree of conjugation (e.g. theidentity of the non-polypeptide moiety, the number of non-polypeptidemoieties desirable or possible to conjugate to the polypeptide, wherethey should be conjugated or where conjugation should be avoided, etc.).For instance, if reduced immunogenicity is desired, the number (andlocation of) attachment groups should be sufficient to shield most orall epitopes. This is normally obtained when a greater proportion of theinterferon β polypeptide is shielded. Effective shielding of epitopes isnormally achieved when the total number of attachment groups availablefor conjugation is in the range of 1-10 attachment groups, in particularin the range of 2-8, such as 3-7.

[0072] Functional in vivo half-life is e.g., dependent on the molecularweight of the conjugate and the number of attachment groups needed forproviding increased half-life thus depends on the molecular weight ofthe non-polypeptide moiety in question. In one embodiment, the conjugateof the invention has a molecular weight of at least 67 kDa, inparticular at least 70 kDa as measured by SDS-PAGE according to Laemmli,U. K., Nature Vol 227 (1970), p680-85. Interferon β has a molecularweight of about 20 kDa, and therefore additional about 50kDa is requiredto obtain the desired effect. This may be, e.g., be provided by 5 10 kDaPEG molecules or as otherwise described herein.

[0073] In order to avoid too much disruption of the structure andfunction of the parent human interferon β molecule the total number ofamino acid residues to be altered in accordance with the presentinvention (as compared to the amino acid sequence shown in SEQ ID NO 2)typically does not exceed 15. Preferably, the interferon β polypeptidecomprises an amino acid sequence, which differs in 1-15 amino acidresidues from the amino acid sequence shown in SEQ ID NO 2, such as in1-8 or in 2-8 amino acid residues, e.g., in 1-5 or in 2-5 amino aresidues from the amino acid sequence shown in SEQ ID NO 2. Thus,normally the interferon β polypeptide comprises an amino acid sequencethat differs from the amino acid sequence shown in SEQ ID NO 2 in 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues.Preferably, the above numbers represent either the total number ofintroduced or the total number of removed amino acid residues comprisingan attachment group for the relevant non-polypeptide moiety, or thetotal number of introduced and removed amino acid residues comprisingsuch group.

[0074] In the conjugate of the invention it is preferred that at leastabout 50% of all conjugatable attachment groups, such as at least about80% and preferably all of such groups are occupied by the relevantnon-polypeptide moiety. Accordingly, in a preferred embodiment theconjugate of the invention comprises, e.g., 1-10 non-polypeptidemoieties, such as 2-8 or 3-6.

[0075] The conjugate of the invention has one or more of the followingimproved properties:

[0076] Reduced immunogenicity as compared to wild-type human interferonβ (e.g. Avonex™ or Rebif®) or to Betaseron®, e.g. a reduction of atleast 25%, such as at least 50%, and more preferably at least 75%;

[0077] Increased functional in vivo half-life and/or increased serumhalf-life as compared to wild-type human interferon β (e.g. Avonex™ orRebif®) or to Betaseron®;

[0078] Reduced or no reaction with neutralizing antibodies from patientstreated with wildtype human interferon β (e.g. Rebif® or Avonex™) orwith Betaseron®, e.g. a reduction of neutralisation of at least 25%,such as of at least 50%, and preferably of at least 75%.

[0079] The magnitude of the antiviral activity of a conjugate of theinvention may not be critical, and thus be reduced (e.g. by up to 75%)or increased (e.g. by at least 5%) or equal to that of wild-type humaninterferon β ((e.g. Avonex™ or Rebif®) or to Betaseron®;

[0080] Furthermore, the degree of antiviral activity as compared toantiproliferative activity of a conjugate of the invention may vary, andthus be higher, lower or equal to that of wildtype human interferon β.

[0081] Conjugate of the Invention, wherein the Non-polypeptide Moiety isa Molecule that has Lysine as an Attachment Group

[0082] In a preferred embodiment the first non-polypeptide moiety haslysine as an attachment group, and thus the interferon β polypeptide isone that comprises an amino acid sequence that differs from that ofwildtype human interferon β in at least one introduced and/or at leastone removed lysine residue. While the non-polypeptide moiety may be anyof those binding to a lysine residue, e.g. the ε-amino group thereof,such as a polymer molecule, a lipophilic group, an organic derivatizingagent or a carbohydrate moiety, it is preferably any of the polymermolecule mentioned in the section entitled “Conjugation to a polymermolecule”, in particular a branched or linear PEG or polyalkylene oxide.Most preferably, the polymer molecule is PEG and the activated moleculeto be used for conjugation is SS-PEG, NPC-PEG, aldehyd-PEG, mPEG-SPA,mPEG-SCM, mPEG-BTC from Shearwater Polymers, Inc, SC-PEG from Enzon,Inc., tresylated mPEG as described in U.S. Pat. No. 5,880,255, oroxycarbonyl-oxy-N -dicarboxyimide-PEG (U.S. Pat. No. 5,122,614).Normally, for conjugation to a lysine residue the non-polypeptide moietyhas a molecular weight of about 5 or 10 kDa.

[0083] In one embodiment the amino acid sequence of the interferon βpolypeptide differs from that of human wildtype interferon β in at leastone removed lysine residue, such as 1-5 removed lysine residues, inparticular 1-4 or 1-3 removed lysine residues. The lysine residue(s) tobe removed, preferably by replacement, is selected from the groupconsisting of K19, K33, K45, K52, K99, K105, K108, K115, K123, K134, andK136. The lysine residue(s) may be replaced with any other amino acidresidue, but is preferably replaced by an arginine or a glutamineresidue in order to give rise to the least structural difference. Inparticular, the polypeptide part may be one, wherein K19, K45, K52and/or K123, preferably K19, K45 and/or K123 has/have been replaced withanother any other amino acid residue, preferably arginine or glutamine.For instance, the interferon β polypeptide part of a conjugate of theinvention comprises a combination of amino acid substitutions selectedfrom the following list:

[0084] K19R+K45R+K123R;

[0085] K19Q+K45R+K123R;

[0086] K19R+K45Q+K123R;

[0087] K19R+K45R+K123Q;

[0088] K19Q+K45Q+K123R;

[0089] K19R+K45Q+K123Q;

[0090] K19Q+K45R+K123Q;

[0091] K19Q+K45Q+K123Q;

[0092] K45R+K123R;

[0093] K45Q+K123R;

[0094] K45Q+K123Q;

[0095] K45R+K123Q;

[0096] K19R+K123R;

[0097] K19Q+K123R;

[0098] K19R+K123Q;

[0099] K19Q+K123Q;

[0100] K19R+K45R;

[0101] K19Q+K45R;

[0102] K19R+K45Q; or

[0103] K19Q+K45Q.

[0104] In addition or alternatively to the amino acid substitutionsmentioned in the above list the polypeptide part may comprise at leastone substitution selected from the group consisting of K33R, K33Q, K52R,K52Q, K99R, K99Q, K105R, K105Q, K108R, K108Q, K115R, K115Q, K134R,K134Q, K136R, and K136Q, e.g. at least one of the followingsubstitutions:

[0105] K52R+K134R;

[0106] K99R+K136R;

[0107] K33R+K105R+K136R;

[0108] K52R+K108R+K134R;

[0109] K99R+K115R+K136R;

[0110] K19R+K33R+K45R+K123R;

[0111] K19R+K45R+K52R+K123R;

[0112] K19R+K33R+K45R+K52R+K123R; or

[0113] K19R+K45R+K52R+K99R+K123R.

[0114] In a further embodiment the the amino acid sequence of theinterferon β polypeptide differs from that shown in SEQ ID NO 2 in thata lysine residue has been introduced by substitution of at least oneamino acid residue occupying a position that in the parent interferon βmolecule is occupied by a surface exposed amino acid residue, preferablyan amino acid residue having at least 25%, such as at least 50% of itsside chain exposed to the surface. Preferably, the amino acid residue tobe substituted is selected from the group consisting of N4, F8, L9, R11,S12, F15, Q16, Q18, L20, W22, Q23, G26, R27, L28, E29, Y29, L32, R35,M36, N37, D39, P41, E42, E43, L47, Q48, Q49, T58, Q64, N65, F67, A68,R71, Q72, D73, S75, S76, G78, N80, E81, I83, E85, N86, A89, N90, Y92,H93, H97, T100, L102, E103, L106, E107, E109, D110, F111, R113, G114,L116, M117, L120, H121, R124, G127, R128, L130, H131, E137, Y138, H140,I145, R147, V148, E149, R152, Y155, F156, N158, R159, G162, Y163, R165and N166 of SEQ ID NO 2.

[0115] More preferably, the amino acid sequence of the interferon βpolypeptide differs from the amino acid sequence shown in SEQ ID NO 2 inthat a lysine residue has been introduced, by substitution, of at leastone amino acid residue occupying a position selected from the groupconsisting of N4, F8, L9, R11, S12, G26, R27, E29, R35, N37, D39, E42,L47, Q48, Q49, A68, R71, Q72, D73, S75, G78, N80, E85, N86, A89, Y92,H93, D110, F111, R113, L116, H121, R124, G127, R128, R147, V148, Y155,N158, R159, G162 and R165, even more preferably selected from the groupconsisting of N4, R11, G26, R27, Q48, Q49, R71, D73, S75, N80, E85, A89,Y92, H93, F111, R113, L116, R124, G127, R128, Y155, N158, and G162, andmost preferably selected from the group consisting of R11, Q49, R71,S75, N80, E85, A89, H93, F111, R113, L116 and Y155, and most preferablyQ49 and F111.

[0116] In accordance with this embodiment, the interferon β polypeptidecomprises a substitution to lysine in one or more of the abovepositions, in particular in 1-15, such as 1-8 or 1-5, and preferably inat least two positions, such as 2-8 or 2-5 positions.

[0117] In a further embodiment the amino acid sequence of the interferonβ polypeptide part of a conjugate differs in at least one removed and atleast one introduced lysine residue, such as 1-5 or 2-5 removed lysineresidues and 1-5 or 2-5 introduced lysine residues. It will beunderstood that the lysine residues to be removed and introducedpreferably are selected from those described in the present section.

[0118] In accordance with this embodiment of the invention, the totalnumber of conjugatable lysine residues is preferably in the range of1-10, such as 2-8 or 3-7.

[0119] For instance, the interferon β polypeptide part of the conjugateaccording to this embodiment may comprise at least one of the followingsubstitutions: R11K, Q48K, Q49K, R71K, S75K, N80K, E85K, A89K, H93K,F111K, R113K, L116K and Y155K; more preferably R11K, Q49K, R71K, S75K,N80K, E85K, A89K, H93K, F111K, R113K, L116K and Y115K, in combinationwith at least one of the substitutions: K19R/Q K33R/Q K45R/Q, K52R/Q,K99R/Q, K105R/Q, K108R/Q, K115R/Q, K123R/Q, K134R/Q, and K136R/Q,wherein R/Q indicates substitution to an R or a Q residue, preferably anR residue. More preferably, the interferon β polypeptide comprises atleast one of the following substitutions R11K, Q49K, R71K, S75K, N80K,E85K, A89K, H93K, F111K, R113K, L116K and Y155K, in particular Q49K,F111,K and/or N80K, in combination with substitution of at least one ofK19, K45, K52 and/or K123, preferably to an R or a Q residue. Inparticular, the interferon β polypeptide comprises at least one of thesubstitutions Q49K, F111K and N80K in combination with at least one ofthe substitutions mentioned above for removal of a lysine residue. Forinstance, the interferon β polypeptide may comprise the followingsubstitutions:

[0120] Y+Z+K19R+K45R+K123R;

[0121] Y+Z+K19Q+K45R+K123R;

[0122] Y+Z+K19R+K45Q+K123R;

[0123] Y+Z+K19R+K45R+K123Q;

[0124] Y+Z+K19Q+K45Q+K123R;

[0125] Y+Z+K19R+K45Q+K123Q;

[0126] Y+Z+K19Q+K45R+K123Q;

[0127] Y+Z+K19Q+K45Q+K123Q;

[0128] Y+Z+K45R+K123R;

[0129] Y+Z+K45Q+K123R;

[0130] Y+Z+K45Q+K123Q;

[0131] Y+Z+K45R+K123Q;

[0132] Y+Z+K19R+K123R;

[0133] Y+Z+K19Q+K123R;

[0134] Y+Z+K19R+K123Q;

[0135] Y+Z+K19Q+K123Q;

[0136] Y+Z+K19R+K45R;

[0137] Y+Z+K19Q+K45R;

[0138] Y+Z+K19R+K45Q; or

[0139] Y+Z+K19Q+K45Q, wherein Y is selected from the group of Q49K,F111K, N80K, Q49K+F111K, Q49K+N80K, F111K+N80K and Q49K+F111K+N80K and Zis absent or comprises at least one substitution selected from the groupconsisting of K33R, K33Q, K52R, K52Q, K99R, K99Q, K105R, K105Q, K108R,K108Q, K115R, K115Q, K134R, K134Q, K136R, and K136Q. Preferably, theinterferon β polypeptide comprises the following substitutionY+Z+K19R+K45Q+K123R, wherein Y and Z have the above meaning.

[0140] More specifically, according to this embodiment the interferon βpolypeptide may comprise one of the following substitutions:

[0141] K19R+K45R+F111K+K123R;

[0142] K19R+K45R+Q49K+F111K+K123R;

[0143] K19R+K45R+Q49K+K123R;

[0144] K19R+K45R+F111K;

[0145] K19R+K45R+Q49K+F111K;

[0146] K19R+Q49K+K123R;

[0147] K19R+Q49K+F111K+K123R;

[0148] K45Q+F111K+K123Q;

[0149] K45R+Q49K+K123R; or

[0150] K45R+Q49K+F111K+K123R.

[0151] Especially for expression in a non-glycosylating host such as E.coli the interferon β polypeptide may contain the substitution N80K orC17S+N80K, optionally in combination with one or more of K19R/Q; K45R/Q;K52R/Q or K123R/Q. The substitution N80K is of particular interest, whenthe interferon β polypeptide is expressed in a non-glycosylating hostcell, since N80 constitutes part of an inherent glycosylation site ofhuman interferon β and conjugation at such site may mimick naturalglycosylation.

[0152] Furthermore, it is preferred that the conjugate according to thisaspect comprises at least two first non-polypeptide moieties, such as2-8 moieties.

[0153] Conjugate of the Invention wherein the Non-polypeptide MoietyBinds to a Cysteine Residue

[0154] In a still further aspect, the invention relates a conjugateexhibiting interferon β activity and comprising at least one firstnon-polypeptide conjugated to at least one cysteine residue of aninterferon β polypeptide, the amino acid sequence of which differs fromthat of wildtype human interferon β in that at least one cysteineresidue has been introduced, preferably by substitution, into a positionthat in the parent interferon β molecule is occupied by an amino acidresidue that is exposed to the surface of the molecule, preferably onethat has at least 25%, such as at least 50% of its side chain exposed tothe surface. For instance, the amino acid residue is selected from thegroup consisting of F8, L9, R11, S12, F15, Q16, Q18, L20, W22, L28, L32,M36, P41, T58, Q64, N65, F67, I83, E85, N86, A89, N90, Y92, H93, H97,T100, L102, E103, L106, M117, L120, H121, R124, G127, R128, L130, H131,H140, I145, R147, V148, E149, R152, Y155, and F156 of SEQ ID NO 2.

[0155] Additionally or alternatively, the substitution is preferablyperformed at a position occupied by a threonine or serine residue. Forinstance, such position is selected from the group consisting of S2,S12, S13, T58, S74, S75, S76, T77, T82, T100, T112, S118, S119, S139,T144, and T161, more preferably S2, S12, S13, S74, S75, S76, T77, T82,T100, T112, S118, S119, S139, and T144 (side chain surface exposed),still more preferably S2, S12, S75, S76, T82, T100, S119 and S139 (atleast 25% of its side chain exposed), and even more preferably S12, S75,T82 and T100 (at least 50% of its side chain exposed). Of the abovethreonine or serine substitutions, serine substitutions are preferred.Accordingly, in even more preferred embodiments, the position isselected from the group consisting of S2, S12, S13, S74, S75, S76, S118,S119 and S139, more preferably S2, S12, S13, S74, S75, S76, S118, S119and S139, even more preferably S2, S12, S75, S76, S119 and S139, andstill more preferably S12 and S75.

[0156] In one embodiment, only one cysteine residue is introduced intothe interferon β polypeptide in order to avoid formation of disulphidebridges between two or more introduced cysteine residues. In thisconnection C17 present in wildtype human interferon β may be removed,preferably by substitution, in particular by substitution with S or A.In another embodiment, two or more cysteine residues are introduced,such as 2-6 or 2-4 cysteine residues. Preferably, the interferon βpolypeptide part of the conjugate according to this embodiment of theinvention comprises the mutation L47C, Q48C, Q49C, D111C, F111C orR113C, in particular only one of these mutations, optionally incombination with the mutation C17S. Also, the interferon β polypeptidemay comprise the substitution C17S+N80C.

[0157] While the first non-polypeptide moiety according to this aspectof the invention may be any molecule which, when using the givenconjugation method has cysteine as an attachment group (such as acarbohydrate moiety, a lipophilic group or an organic derivatizingagent), it is preferred that the non-polypeptide moiety is a polymermolecule. The polymer molecule may be any of the molecules mentioned inthe section entitled “Conjugation to a polymer molecule”, but ispreferably selected from the group consisting of linear or branchedpolyethylene glycol or polyalkylene oxide. Most preferably, the polymermolecule is VS-PEG. The conjugation between the polypeptide and thepolymer may be achieved in any suitable manner, e.g. as described in thesection entitled “Conjugation to a polymer molecule”, e.g. in using aone step method or in the stepwise manner referred to in said section.When the interferon β polypeptide comprises only one conjugatablecysteine residue, this is preferably conjugated to a firstnon-polypeptide moiety with a molecular weight of at least 20 kDa,either directly conjugated or indirectly through a low molecular weightpolymer (as disclosed in WO 99/55377). When the conjugate comprises twoor more first non-polypeptide moieties, normally each of these has amolecular weight of 5 or 10 kDa.

[0158] Conjugate of the Invention wherein the Non-polypeptide MoietyBinds to an Acid Group

[0159] In a still further aspect the invention relates to a conjugateexhibiting interferon β activity and comprising at least one firstnon-polypeptide moiety having an acid group as the attachment group,which moiety is conjugated to at least one aspartic acid residue or oneglutamic acid residue of an interferon β polypeptide, the amino acidsequence of which differs from that of wildtype human interferon β in atleast one introduced and/or at least one removed aspartic acid orglutamic acid residue, respectively. The relevant amino acid residue maybe introduced in any position occupied by a surface exposed amino.acidresidue, preferably by an amino acid residue having more than 25% of itsside chain surface exposed. Preferably, at least one amino acid residueoccupying a position selected from the group consisting of N4, L5, L6,F8, L9, Q10, R11, S12, S13, F15, Q16, Q18, K19, L20, W22, Q23, L24, N25,G26, R27, Y30, M36, Q46, Q48, Q49, I66, F67, A68, I69, F70, R71, S75,T82, I83, L87, A89, N90, V91, Y92, H93, Q94, I95, N96, H97, K108, F111,L116, L120, K123, R124, Y126, G127, R128, L130, H131, Y132, K134, A135,H140, T144, R147, Y155, F156, N158, R159, G162, Y163 and R165 has beensubstituted with an aspartic acid residue or a glutamic acid residue.

[0160] More preferably, the position is selected from the groupconsisting of N4, L5, F8, L9, R11, S12, F15, Q16, Q18, K19, W22, Q23,G26, R27, Y30, M36, Q46, Q48, Q49, A68, R71, S75, T82, A89, N90, Y92,H93, N96, H97, K108, F111, L116, L120, K123, R124, G127, R128, L130,H131, K134, A135, H140, Y155, N158, R159, G162, Y163 and R165, such asfrom the group consisting of N4, L5, F8, S12, F15, Q16, K19, W22, Q23,R27, Y30, M36, Q46, Q48, Q49, R71, S75, T82, A89, Y92, H93, K108, F111,L116, K123, R124, G127, H131, K134, A135, Y155 and R165, still morepreferably from the group consisting of N4, L5, F8, S12, F15, Q16, K19,W22, Q23, R27, Y30, Q46, Q48, Q49, S75, T82, A89, Y92, H93, K108, F111,L116, R124, G127, H131, K134, Y155 and R165, such as from the groupconsisting of L5, F8, S12, F15, Q16, K19, W22, Q23, Q48, Q49, Y92, H93,R124, G127, H131 and Y155, even more preferably from the groupconsisting of S12, Q16, K19, Q23, Q48, Q49, Y92, H93, R124, G127, H131and Y155, such as from the group consisting of S12, Q16, K19, Q23, Q48,Y92, H93, R124, G127, H131 and Y155, in particular from the groupconsisting of S12, Q16, K19, Q23, Q48, H93 and H131, even morepreferably from the group consisting of S12, Q16, K19, Q48, H93 andH131, and most preferably from the group consisting of Q16 and Q48.

[0161] Furthermore, in order to obtain a sufficient number ofnon-polypeptide moieties it is preferred that that least two asparticacid residues or at least two glutamic acid residues be introduced,preferably in two positions selected from any of the above lists. Also,it is preferred that the conjugate according to this aspect comprises atleast two first non-polypeptide moieties.

[0162] In case of removal of an amino acid residue, the amino acidsequence of the interferon β polypeptide differs from that of humanwildtype interferon β in at least one removed aspartic acid or glutamicacid residue, such as 1-5 removed residues, in particular 1-4 or 1-3removed aspartic acid or glutamic acid residues. The residue(s) to beremoved, preferably by replacement, is selected from the groupconsisting of D34, D39, D54, D73, D110, E29, E42, E43, E53, E61, E81,E85, E103, E104, E107, E109, E137 and E149. The aspartic glutamic acidresidue(s) may be replaced with any other amino acid residue, but ispreferably replaced by an arginine or a glutamine residue.firstnon-polypeptide moiety can be any non-polypeptide moiety with suchproperty, it is presently preferred that the non-polypeptide moiety is apolymer molecule or an organic derivatizing agent having an acid groupas an attachment group, in particular a polymer molecule such as PEG,and the conjugate is prepared, e.g., as described by Sakane andPardridge, Pharmceutical Research, Vol. 14, No. 8, 1997, pp 1085-1091.Normally, for conjugation to an acid group the non-polypeptide moietyhas a molecular weight of about 5 or 10 kDa.

[0163] Conjugate of the Invention Comprising a Second Non-polypeptideMoiety

[0164] In addition to a first non-polypeptide moiety (as described inthe preceding sections), the conjugate of the invention may comprise asecond non-polypeptide moiety of a different type as compared to thefirst non-polypeptide moiety. Preferably, in any of the above describedconjugates wherein the first non-polypeptide moiety is, e.g., a polymermolecule such as PEG, a second non-polypeptide moiety is a sugar moiety,in particular an N-linked sugar moiety. While the second non-polypeptidemoiety may be attached to a natural glycosylation site of humaninterferon β, e.g. the N-linked glycosylation site defined by N80, it isnormally advantageous to introduce at least one additional glycosylationsite in the interferon β polypeptide. Such site is e.g. any of thosedescribed in the immediately preceding section entitled “Conjugate ofthe invention wherein the non-polypeptide moiety is a sugar moiety”.Furthermore, in case at least one additional glycosylation site isintroduced this may be accompanied by removal of an existingglycosylation site as described below.

[0165] It will be understood that in order to obtain an optimaldistribution of attached first and second non-polypeptide moieties, theinterferon β polypeptide may be modified in the number and distributionof attachment groups for the first as well as the second non-polypeptidemoiety so as to have e.g. at least one removed attachment group for thefirst non-polypeptide moiety and at least one introduced attachmentgroup for the second non-polypeptide moiety or vice versa. For instance,the interferon β polypeptide comprises at least two (e.g. 2-5) removedattachment groups for the first non-polypeptide moiety and at least one(e.g. 1-5) introduced attachment groups for the second non-polypeptidemoiety or vice versa. Of particular interest is a conjugate wherein thefirst non-polypeptide moiety is a polymer molecule such as PEG havinglysine as an attachment group, and the second non-polypeptide moiety isan N-linked sugar moiety.

[0166] More specifically, the conjugate of the invention may be oneexhibiting interferon β activity and comprising at least one polymermolecule, preferably PEG, and at least one sugar moiety covalentlyattached to an interferon β polypeptide, the amino acid sequence ofwhich differs from that of wild-type human interferon β in

[0167] a) at least one introduced and/or at least one removed amino acidresidue comprising an attachment group for the polymer molecule; and

[0168] b) at least one introduced and/or at least one removed in vivoglycosylation site, in particular an N-glycosylation site,

[0169] provided that when the attachment group for the polymer moleculeis a cysteine residue, and the sugar moiety is an N-linked sugar moiety,a cysteine residue is not inserted in such a manner that anN-glycosylation site is destroyed. WO 99/03887 suggests that a cysteineresidue can be introduced into the natural N-glycosylation site ofinterferon β.

[0170] In a specific embodiment, the interferon β polypeptide comprisesone of the following sets of mutations:

[0171] K19R+K45R+Q49N+Q51T+F111N+R113T+K123R;

[0172] K19R+K45R+Q49N+Q51T+F111N+R113T; or

[0173] K19R+K45R+Q49N+Q51T+K123R.

[0174] Conjugate of the Invention wherein the Non-polypeptide Moiety isa Sugar Moiety

[0175] When the conjugate of the invention comprises at least one sugarmoiety attached to an in vivo glycosylation site, in particular anN-glycosylation site, this is either the natural N-glycosylation site ofwild-type human interferon β at position N80, i.e. defined by amino acidresidues N80, E81, T82 and 83, or a new in vivo glycosylation siteintroduced into the interferon β polypeptide. The in vivo glycosylationsite may be an O-glycosylation site, but is preferably anN-glycosylation site.

[0176] More specifically, in one aspect the invention relates to aconjugate exhibiting interferon β activity and comprising an interferonβ polypeptide, the amino acid sequence of which differs from that ofwild-type human interferon β in at least one introduced glycosylationsite, the conjugate further comprising at least one un-PEGylated sugarmoiety attached to an introduced glycosylation site.

[0177] In another aspect the invention relates to a conjugate exhibitinginterferon β activity and comprising an interferon β polypeptide, theamino acid sequence of which differs from that of wild-type humaninterferon β in that a glycosylation site has been introduced orremoved, provided that if only a glycosylation site is removed (and thusthat no glycosylation site is introduced) the interferon β polypeptidedoes not comprise one or more of the following substutions: N80C, E81Cor T82C. The latter substitution is suggested in WO 99/03887.

[0178] For instance, an in vivo glycosylation site is introduced into aposition of the parent interferon β molecule occupied by an amino acidresidue exposed to the surface of the molecule, preferably with morethan 25% of the side chain exposed to the solvent, in particular morethan 50% exposed to the solvent (these positions are identified in theMethods section herein). The N-glycosylation site is introduced in sucha way that the N-residue of said site is located in said position.Analogously, an O-glycosylation site is introduced so that the S or Tresidue making up such site is located in said position. Furthermore, inorder to ensure efficient glycosylation it is preferred that the in vivoglycosylation site, in particular the N residue of the N-glycosylationsite or the S or T residue of the O-glycosylation site, is locatedwithin the first 141 amino acid residues of the interferon βpolypeptide, more preferably within the first 116 amino acid residues.Still more preferably, the in vivo glycosylation site is introduced intoa position wherein only one mutation is required to create the site(i.e. where any other amino acid residues required for creating afunctional glycosylation site is already present in the molecule).

[0179] Substitutions that lead to introduction of an additionalN-glycosylation site at positions exposed at the surface of theinterferon β molecule and occupied by amino acid residues having morethan 25% of the side chain exposed to the surface include:

[0180] S2N+N4S/T, L6S/T, L5N+G7S/T, F8N+Q10S/T, L9N+R11S/T, R11N,R11N+S13T, S12N+N14S/T, F15N+C17S/T, Q16N+Q18S/T, Q18N+L20S/T,K19N+L21S/T, W22N+L24S/T, Q23N+H25S/T, G26N+L28S/T, R27N+E29S/T,L28S+Y30S/T, Y30N+L32S/T, L32N+D34S/T, K33N+R35S/T, R35N+N37S/T,M36N+F38S/T, D39S/T, D39N+P41S/T, E42N+I44S/T, Q43N+K45S/T, K45N+L47S/T,Q46N+Q48S/T, L47N+Q49T/S, Q48N+F50S/T, Q49N+Q51S/T, Q51N+E53S/T,K52N+D54S/T, L57N+I59S/T, Q64N+I66S/T, A68N+F70S/T, R71N+D73S/T, Q72N,Q72N+S74T, D73N, D73N+S75T, S75N+T77S, S75N, S76N+G78S/T, E81N+I83S/T,T82N+V84S/T, E85N+L87S/T, L88S/T, A89N+V91S/T, Y92S/T, Y92N+Q94S/T,H93N+I95S/T, L98S/T, H97N+K99S/T, K99N+V101S/T, T100N+L102S/T,E103N+K105S/T, E104N+L106S/T, K105N+E107S/T, E107N+E109S/T,K108N+D110S/T, E109N+F111S/T, D110N+T112S, D110N, F111N+R113S/T,R113N+K115S/T, G114N+L116S/T, K115N+M117S/T, L116N, L116N+S118T,S119N+H212S/T, L120N+L122S/T, H121N+K123S/T, K123N+Y125S/T,R124N+Y126S/T, G127N+I129S/T, R128N+L130S/T, L130N+Y132S/T,H131N+L133S/T, K134N+K136S/T, A135N+E137S/T, K136N+Y138S/T, E137N,Y138N+H140S/T, H140N+A142S/T, V148N+I150S/T, R152N+F154S/T,Y155N+I157S/T, L160S/T, R159N+T161S, R159N, G162N+L164S/T, andY163N+R165S/T.

[0181] Substitutions that lead to introduction of an additionalN-glycosylation site at positions exposed at the surface of theinterferon β molecule having more than 50% of the side chain exposed tothe surface include: L6S/T, L5N+G7S/T, F8N+Q10S/T, L9N+R11S/T,S12N+N14S/T, F15N+C17S/T, Q16N+Q18S/T, K19N+L21S/T, W22N+L24S/T,Q23N+H25S/T, G26N+L28S/T, R27N+E29S/T, Y30N+L32S/T, K33N+R35S/T,R35N+N37S/T, M36N+F38S/T, D39S/T, D39N+P41S/T, E42N+I44S/T, Q46N+Q48S/T,Q48N+F50S/T, Q49N+Q51S/T, Q51N+E53S/T, K52N+D54S/T, L57N+I59S/T,R71N+D73S/T, D73N, D73N+S75T, S75N+T77S, S75N, S76N+G78S/T, E81N+I83S/T,T82N+V84S/T, E85N+L87S/T, A89N+V91S/T, Y92S/T, Y92N+Q94S/T, H93N+I95S/T,T100N+L102S/T, E103N+K105S/T, E104N+L106S/T, E107N+E109S/T,K108N+D110S/T, D110N+T112S, D110N, F111N+R113S/T, R113N+K115S/T, L116N,L116N+S118T, K123N+Y125S/T, R124N+Y126S/T, G127N+I129S/T, H131N+L133S/T,K134N+K136S/T, A135N+E137S/T, E137N, V148N+I150S/T, and Y155N+I157S/T.

[0182] Among the substitutions mentioned in the above lists, those arepreferred that have the N residue introduced among the 141 N-terminalamino acid residues, in particular among the 116 N-terminal amino acidresidues.

[0183] Substitutions that lead to introduction of an N-glycosylationsite by only one amino acid substitution include: L6S/T, R11N, D39S/T,Q72N, D73N, S75N, L88S/T, Y92S/T, L98S/T, D110N, L116N, E137N, R159N andL160S/T. Among these, a substitution is preferred that is selected fromthe group consisting of L6S/T, R11N, D39S/T, Q72N, D73N, S75N, L88S/T,Y92S/T, L98S/T, D110N and L116N, more preferably from the groupconsisting of L6S/T, D39S/T, D73N, S75N, L88S/T, D110N, L116N and E137N;and most preferably selected from the group consisting of L6S/T, D39S/T,D73N, S75N, L88S/T, D110N and L116N.

[0184] One espcecially preferred interferon β polypeptide according tothis aspect include at least one of the following substitutions:

[0185] S2N+N4T/S, L9N+R11T/S, R11N, S12N+N14T/S, F15N+C17S/T,Q16N+Q18T/S, K19N+L21T/S, Q23N+H25T/S, G26N+L28T/S, R27N+E29T/S,L28N+Y30T/S, D39T/S, K45N+L47T/S, Q46N+Q48T/S, Q48N+F50T/S, Q49N+Q51T/S,Q51N+E53T/S, R71N+D73T/S, Q72N, D73N, S75N, S76N+G78T/S, L88T/S, Y92T/S,N93N+195T/S, L98T/S, E103N+K105T/S, E104N+L106T/S, E107N+E109T/S,K108N+D110T/S, D110N, F111N+R113T/S, or L116N, more preferably at leastone of the following substitutions: S2N+N4T, L9N+R11T, 49N+Q51T orF111N+R113T or R71N+D73T, in particular 49N+Q51T or F111N+R113T orR71N+D73T. For instance, the interferon β polypeptide comprises one ofthe following sets of substitutions:

[0186] Q49N+Q51T+F111N+R113T;

[0187] Q49N+Q51T+R71N+D73T+F111N+R113T;

[0188] S2N+N4T+F111N+R113T;

[0189] S2N+N4T+Q49N+Q51T;

[0190] S2N+N4T+Q49N+Q51T+F111N+R113T;

[0191] S2N+N4T+L9N+R11T+Q49N+Q51T;

[0192] S2N+N4T+L9N+R11T+F111N+R113T;

[0193] S2N+N4T+L9N+R11T+Q49N+Q51 T+F111N+R113T;

[0194] L9N+R11T+Q49N+Q51T;

[0195] L9N+R11T+Q49N+Q51T+F111N+R113T; or

[0196] L9N+R11T+F111N+R113T

[0197] It will be understood that in order to introduce a functional invivo glycosylation site the amino acid residue inbetween the N-residueand the S/T residue is different from proline. Normally, the amino acidresidue inbetween will be that occupying the relevant position in theamino acid sequence shown in SEQ ID NO 2. For instance, in thepolypeptide comprising the substitutions Q49N+Q51S, position 50 is theposition inbetween.

[0198] The interferon β polypeptide part of a conjugate of the inventionmay contain a single in vivo glycosylation site. However, in order toobtain efficient shielding of epitopes present on the surface of theparent polypeptide it is often desirable that the polypeptide comprisesmore than one in vivo glycosylation site, in particular 2-7 in vivoglycosylation sites, such as 2, 3, 4, 5, 6 or 7 in vivo glycosylationsites. Thus, the interferon β polypeptide may comprise one additionalglycosylation site, or may comprise two, three, four, five, six, sevenor more introduced in vivo glycosylation sites, preferably introduced byone or more substitutions described in any of the above lists.

[0199] As indicated above, in addition to one or more introducedglycosylation sites, existing glycosylation sites may have been removedfrom the interferon β polypeptide. For instance, any of the above listedsubstitutions to introduce a glycosylation site may be combined with asubstitution to remove the natural N-glycosylation site of humanwild-type interferon β. For instance, the interferon β polypeptide maycomprise a substitution of N80, e.g. one of the substitutionsN80K/C/D/E, when a first non-polypeptide polypeptide is one having oneof K, C, D, E as an attachment group. For instance, the interferon βpolypeptide may comprise at least one of the following substitutions:S2N+N4T/S, L9N+R11T/S, R11N, S12N+N14T/S, F15N+C17S/T, Q16N+Q18T/S,K19N+L21T/S, Q23N+H25T/S, G26N+L28T/S, R27N+E29T/S, L28N+Y30T/S, D39T/S,K45N+L47T/S, Q46N+Q48T/S, Q48N+F50T/S, Q49N+Q51T/S, Q51N+E53T/S,R71N+D73T/S, Q72N, D73N, S75N, S76N+G78T/S, L88T/S, Y92T/S, N93N+195T/S,L98T/S, E103N+K105T/S, E104N+L106T/S, E107N+E109T/S, K108N+D110T/S,D110N, F111N+R113T/S, or L116N in combination with N80K/C/D/E. Morespecifically, the interferon β polypeptide may comprise thesubstitution: Q49N+Q51T or F111N+R113T or R71N+D73T, in particularQ49N+Q51T+F111N+R113T or Q49N+Q51T+R71N+D73T+F111N+R,13T, in combinationwith N80K/C/D/E.

[0200] Any of the glycosylated variants disclosed in the present sectionhaving introduced and/or removed at least one glycosylation site, suchas the variant comprising the substitutions Q48N+F50T/S,Q48N+F50T/S+F111N+R113T/S, Q49N+Q51T/S, F111N+R113T/S, orQ49N+Q51T/S+F111N+R113T/S, may further be conjugated to a polymermolecule, such as PEG, or any other non-polypeptide moiety. For thispurpose the conjugation may be achieved by use of attachment groupsalready present in the interferon β polypeptide or attachment groups mayhave been introduced and/or removed, in particular such that a total of1-6, in particular 3-4 or 1, 2, 3, 4, 5, or 6 attachment groups areavailable for conjugation. Preferably, in a conjugate of the inventionwherein the interferon β polypeptide comprises two glycosylation sites,the number and molecular weight of the non-polypeptide moiety is chosenso as that the total molecular weight added by the non-polypeptidemoiety is in the range of 20-40 kDa, in particular about 20 kDa or 30kDa.

[0201] In particular, the glycosylated variant may be conjugated to anon-polypeptide moiety via a lysine attachment group, and one or morelysine residues of the parent polypeptide may have been removed, e.g. byany of the substitutions mentioned in the section entitled “Conjugate ofthe invention, wherein the non-polypeptide moiety is a molecule whichhas lysine as an attachment group”, in particular the substitutionsK19R+K45R+K123R. Alternatively or additionally, a lysine residue mayhave been introduced, e.g. by any of the substitutions mentioned in saidsection, in particular the substitution R71K. Accordingly, one specificconjugate of the invention is one, which comprises a glycosylatedinterferon β polypeptide comprising the mutations Q49N+Q51T+F111N+R113T+K19R+K45R+K123R orQ49N+Q51T+F111N+R113T+K19R+K45R+K123R+R71K further conjugated to PEG.The glycosylated polypeptide part of said conjugate is favourablyproduced in CHO cells and PEGylated subsequent to purification usinge.g. SS-PEG, NPC-PEG, aldehyd-PEG, mPEG-SPA, mPEG-SCM, mPEG-BTC fromShearwater Polymers, Inc, SC-PEG from Enzon, Inc., tresylated mPEG asdescribed in U.S. Pat. No. 5,880,255, oroxycarbonyl-oxy-N-dicarboxyimide-PEG (U.S. Pat. No. 5,122,614).

[0202] Alternatively, to PEGylation via a lysine group, the glycosylatedconjugate according to this embodiment may be PEGylated via a cysteinegroup as described in the section entitled “Conjugate of the invention,wherein the non-polypeptide moiety is a molecule that has cysteine as anattachment group” (for this purpose the interferon β polypeptide may,e.g. comprising at least one of the mutations N80C, R71C and C17S), viaan acid group as described in the section entitled “Conjugation of theinvention wherein the non-polypeptide moiety binds to an acid group”, orvia any other suitable group.

[0203] Other Conjugates of the Invention

[0204] In addition to the introduction and/or removal of amino acidresidues comprising an attachment group for the non-polypeptide moietyof choice (as described in any of the sections above entitled “Conjugateof the invention . . . ”) the interferon β polypeptide part of theconjugate may contain further substitutions. A preferred example is asubstitution of any of the residues, M1, C17, N80 or V101, e.g. one ormore of the following substitutions: C17S; N80K/C/D/E; V101Y/W/F/,H; adeletion of M1; or M1K. The substitution M1K is of particular interestwhen the interferon β polypeptide is expressed with a tag, e.g. aHis-14tag, where such tag is to be removed by DAP (diaminopeptidase)subsequent to purification and/or conjugation.

[0205] Non-polypeptide Moiety of the Conjugate of the Invention

[0206] As indicated further above the non-polypeptide moiety of theconjugate of the invention is preferably selected from the groupconsisting of a polymer molecule, a lipophilic compound, a sugar moiety(by way of in vivo glycosylation) and an organic derivatizing agent. Allof these agents may confer desirable properties to the polypeptide partof the conjugate, in particular reduced immunogenicity and/or increasedfunctional in vivo half-life and/or increased serum half-life. Thepolypeptide part of the conjugate may be conjugated to only one type ofnon-polypeptide moiety, but may also be conjugated to two or moredifferent types of non-polypeptide moieties, e.g. to a polymer moleculeand a sugar moiety, to a lipophilic group and a sugar moiety, to anorganic derivating agent and a sugar moiety, to a lipophilic group and apolymer molecule, etc. The conjugation to two or more differentnon-polypeptide moieties may be done simultaneous or sequentially. Thechoice of non-polypeptide moiety/ies, e.g. depends on the effect desiredto be achieved by the conjugation. For instance, sugar moieties havebeen found particularly useful for reducing immunogenicity, whereaspolymer molecules such as PEG are of particular use for increasingfunctional in vivo half-life and/or serum half-life. Using a polymermolecule as a first non-polypeptide moiety and a sugar moiety as asecond non-polypeptide moiey may result in reduced immunogenicity andincreased functional in vivo or serum half-life.

[0207] Methods of Preparing a Conjugate of the Invention

[0208] In the following sections “Conjugation to a lipophilic compound”,“Conjugation to a polymer molecule”, “Conjugation to a sugar moiety” and“Conjugation to an organic derivatizing agent” conjugation to specifictypes of non-polypeptide moieties is described.

[0209] Conjugation to a Lipophilic Compound

[0210] For conjugation to a lipophilic compound the followingpolypeptide groups may function as attachment groups: the N-terminal orC-terminal of the polypeptide, the hydroxy groups of the amino acidresidues Ser, Thr or Tyr, the Δ-amino group of Lys, the SH group of Cysor the carboxyl group of Asp and Glu. The polypeptide and the lipophiliccompound may be conjugated to each other, either directly or by use of alinker. The lipophilic compound may be a natural compound such as asaturated or unsaturated fatty acid, a fatty acid diketone, a terpene, aprostaglandin, a vitamine, a carotenoide or steroide, or a syntheticcompound such as a carbon acid, an alcohol, an amine and sulphonic acidwith one or more alkyl-, aryl-, alkenyl- or other multiple unsaturatedcompounds. The conjugation between the polypeptide and the lipophiliccompound, optionally through a linker may be done according to methodsknown in the art, e.g. as described by Bodanszky in Peptide Synthesis,John Wiley, New York, 1976 and in WO 96/12505.

[0211] Conjugation to a Polymer Molecule

[0212] The polymer molecule to be coupled to the polypeptide may be anysuitable polymer molecule, such as a natural or synthetic homo-polymeror heteropolymer, typically with a molecular weight in the range of300-100,000 Da, such as 300-20,000 Da, more preferably in the range of500-10,000 Da, even more preferably in the range of 500-5000 Da.

[0213] Examples of homo-polymers include a polyol (i.e. poly-OH), apolyamine (i.e. poly-NH₂) and a polycarboxylic acid (i.e. poly-COOH). Ahetero-polymer is a polymer, which comprises one or more differentcoupling groups, such as, e.g., a hydroxyl group and an amine group.

[0214] Examples of suitable polymer molecules include polymer moleculesselected from the group consisting of polyalkylene oxide (PAO),including polyalkylene glycol (PAG), such as polyethylene glycol (PEG)and polypropylene glycol (PPG), branched PEGs, poly-vinyl alcohol (PVA),poly-carboxylate, poly-(vinylpyrolidone), polyethylene-co-maleic acidanhydride, polystyrene-co-malic acid anhydride, dextran includingcarboxymethyl-dextran, or any other biopolymer suitable for reducingimmunogenicity and/or increasing functional in vivo half-life and/orserum half-life. Another example of a polymer molecule is human albuminor another abundant plasma protein. Generally, polyalkyleneglycol-derived polymers are biocompatible, non-toxic, non-antigenic,non-immunogenic, have various water solubility properties, and areeasily excreted from living organisms.

[0215] PEG is the preferred polymer molecule to be used, since it hasonly few reactive groups capable of cross-linking compared, e.g., topolysaccharides such as dextran, and the like. In particular,monofunctional PEG, e.g monomethoxypolyethylene glycol (mPEG), is ofinterest since its coupling chemistry is relatively simple (only onereactive group is available for conjugating with attachment groups onthe polypeptide). Consequently, the risk of cross-linking is eliminated,the resulting polypeptide conjugates are more homogeneous and thereaction of the polymer molecules with the polypeptide is easier tocontrol.

[0216] To effect covalent attachment of the polymer molecule(s) to thepolypeptide, the hydroxyl end groups of the polymer molecule must beprovided in activated form, i.e. with reactive functional groups(examples of which include primary amino groups, hydrazide (HZ), thiol,succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide(SSA), succinimidyl proprionate (SPA), succinimidy carboxymethylate(SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS),aldehyde, nitrophenylcarbonate (NPC), and tresylate (TRES)). Suitablyactivated polymer molecules are commercially available, e.g. fromShearwater Polymers, Inc., Huntsville, Ala., USA. Alternatively, thepolymer molecules can be activated by conventional methods known in theart, e.g. as disclosed in WO 90/13540. Specific examples of activatedlinear or branched polymer molecules for use in the present inventionare described in the Shearwater Polymers, Inc. 1997 and 2000 Catalogs(Functionalized Biocompatible Polymers for Research and pharmaceuticals,Polyethylene Glycol and Derivatives, incorporated herein by reference).Specific examples of activated PEG polymers include the following linearPEGs: NHS-PEG (e.g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG,SG-PEG, and SCM-PEG), and NOR-PEG), BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG,CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branchedPEGs such as PEG2-NHS and those disclosed in U.S. Pat. No. 5,932,462 andU.S. Pat. No. 5,643,575, both of which references are incorporatedherein by reference. Furthermore, the following publications,incorporated herein by reference, disclose useful polymer moleculesand/or PEGylation chemistries: U.S. Pat. Nos. 5,824,778, 5,476,653, WO97/32607, EP 229,108, EP 402,378, U.S. Pat. Nos. 4,902,502, 5,281,698,5,122,614, 5,219,564, WO 92/16555, WO 94/04193, WO 94/14758, WO94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924,W095/13090, WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO98/32466, WO 95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO95/13312, EP 921 131, U.S. Pat. No. 5,736,625, WO 98/05363, EP 809 996,U.S. Pat. No. 5,629,384, WO 96/41813, WO 96/07670, U.S. Pat. Nos.5,473,034, 5,516,673, EP 605 963, U.S. Pat. No. 5,382,657, EP 510 356,EP 400 472, EP 183 503 and EP 154 316.

[0217] The conjugation of the polypeptide and the activated polymermolecules is conducted by use of any conventional method, e.g. asdescribed in the following references (which also describe suitablemethods for activation of polymer molecules): Harris and Zalipsky, eds.,Poly(ethylene glycol) Chemistry and Biological Applications, AZC,Washington; R. F. Taylor, (1991), “Protein immobilisation. Fundamentaland applications”, Marcel Dekker, N.Y.; S. S. Wong, (1992), “Chemistryof Protein Conjugation and Crosslinking”, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), “Immobilized Affinity Ligand Techniques”,Academic Press, N.Y.). The skilled person will be aware that theactivation method and/or conjugation chemistry to be used depends on theattachment group(s) of the interferon β polypeptide as well as thefunctional groups of the polymer (e.g. being amino, hydroxyl, carboxyl,aldehyde or sulfydryl). The PEGylation may be directed towardsconjugation to all available attachment groups on the polypeptide (i.e.such attachment groups that are exposed at the surface of thepolypeptide) or may be directed towards specific attachment groups, e.g.the N-terminal amino group (U.S. Pat. No. 5,985,265). Furthermore, theconjugation may be achieved in one step or in a stepwise manner (e.g. asdescribed in WO 99/55377).

[0218] It will be understood that the PEGylation is designed so as toproduce the optimal molecule with respect to the number of PEG moleculesattached, the size and form (e.g. whether they are linear or branched)of such molecules, and where in the polypeptide such molecules areattached. For instance, the molecular weight of the polymer to be usedmay be chosen on the basis of the desired effect to be achieved. Forinstance, if the primary purpose of the conjugation is to achieve aconjugate having a high molecular weight (e.g. to reduce renalclearance) it is usually desirable to conjugate as few high Mw polymermolecules as possible to obtain the desired molecular weight. When ahigh degree of epitope shielding is desirable this may be obtained byuse of a sufficiently high number of low molecular weight polymer (e.g.with a molecular weight of about 5,000 Da) to effectively shield all ormost epitopes of the polypeptide. For instance, 2-8, such as 3-6 suchpolymers may be used.

[0219] In connection with conjugation to only a single attachment groupon the protein (as described in U.S. Pat. No. 5,985,265), it may beadvantageous that the polymer molecule, which may be linear or branched,has a high molecular weight, e.g. about 20 kDa.

[0220] Normally, the polymer conjugation is performed under conditionsaiming at reacting all available polymer attachment groups with polymermolecules. Typically, the molar ratio of activated polymer molecules topolypeptide is 1000-1, in particular 200-1, preferably 100-1, such as10-1 or 5-1 in order to obtain optimal reaction. However, also equimolarratios may be used.

[0221] It is also contemplated according to the invention to couple thepolymer molecules to the polypeptide through a linker. Suitable linkersare well known to the skilled person. A preferred example is cyanuricchloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581;U.S. Pat. No. 4,179,337; Shafer et al., (1986), J. Polym. Sci. Polym.Chem. Ed., 24, 375-378.

[0222] Subsequent to the conjugation residual activated polymermolecules are blocked according to methods known in the art, e.g. byaddition of primary amine to the reaction mixture, and the resultinginactivated polymer molecules removed by a suitable method.

[0223] Covalent in vitro coupling of a carbohydrate moiety to amino acidresidues of interferon β may be used to modify or increase the number orprofile of carbohydrate substituents. Depending on the coupling modeused, the carbohydrate(s) may be attached to a) arginine and histidine(Lundblad and Noyes, Chemical Reagents for Protein Modification, CRCPress Inc. Boca Raton, Fla.), b) free carboxyl groups (e.g. of theC-terminal amino acid residue, asparagine or glutamine), c) freesulfhydryl groups such as that of cysteine, d) free hydroxyl groups suchas those of serine, threonine, tyrosine or hydroxyproline, e) aromaticresidues such as those of phenylalanine or tryptophan or f) the amidegroup of glutamine. These amino acid residues constitute examples ofattachment groups for a carbohydrate moiety, which may be introducedand/or removed in the interferon β polypeptide. Suitable methods of invitro coupling are described in WO 87/05330 and in Aplin etl al., CRCCrit Rev. Biochem., pp. 259-306, 1981. The in vitro coupling ofcarbohydrate moieties or PEG to protein- and peptide-bound Gln-residuescan also be carried out by transglutaminases (T Gases), e.g. asdescribed by Sato et al., 1996 Biochemistry 35, 13072-13080 or in EP725145

[0224] Coupling to a Sugar Moiety

[0225] In order to achieve in vivo glycosylation of an interferon βpolypeptide that has been modified by introduction of one or moreglycosylation sites (see the section “Conjugates of the inventionwherein the non-polypeptide moiety is a sugar moiety”), the nucleotidesequence encoding the polypeptide part of the conjugate must be insertedin a glycosylating, eucaryotic expression host. The expression host cellmay be selected from fungal (filamentous fungal or yeast), insect,mammalian animal cells, from transgenic plant cells or from transgenicanimals. Furthermore, the glycosylation may be achieved in the humanbody when using a nucleotide sequence encoding the polypeptide part of aconjugate of the invention or a polypeptide of the invention in genetherapy. In one embodiment the host cell is a mammalian cell, such as anCHO cell, BHK or HEK cell, e.g. HEK293, or an insect cell, such as anSF9 cell, or a yeast cell, e.g. Saccharomyces cerevisiae, Pichiapastoris or any other suitable glycosylating host, e.g. as describedfurther below. Optionally, sugar moieties attached to the interferon βpolypeptide by in vivo glycosylation are further modified by use ofglycosyltransferases, e.g. using the glyco Advance™ technology marketedby Neose, Horsham, Pa., USA. Thereby, it is possible to, e.g., increasethe sialyation of the glycosylated interferon β polypeptide followingexpression and in vivo glycosylation by CHO cells.

[0226] Coupling to an Organic Derivatizing Agent

[0227] Covalent modification of the interferon β polypeptide may beperformed by reacting (an) attachment group(s) of the polypeptide withan organic derivatizing agent. Suitable derivatizing agents and methodsare well known in the art. For example, cysteinyl residues most commonlyare reacted with α-haloacetates (and corresponding amines), such aschloroacetic acid or chloroacetamide, to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteinyl residues also are derivatizedby reaction with bromotrifluoroacetone, α-bromo-β-(4-imidozoyl)propionicacid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyldisulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.Histidyl residues are derivatized by reaction withdiethylpyrocarbonateat pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful; the reaction is preferably performed in 0.1 M sodium cacodylateat pH 6.0. Lysinyl and amino terminal residues are reacted with succinicor other carboxylic acid anhydrides. Derivatization with these agentshas the effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate. Arginyl residues are modified by reaction with one orseveral conventional reagents, among them phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginine residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas the arginine guanidino group. Carboxyl side groups (aspartyl orglutamyl or C-terminal amino acid residue) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

[0228] Blocking of Functional Site

[0229] It has been reported that excessive polymer conjugation can leadto a loss of activity of the interferon β polypeptide to which thepolymer is conjugated. This problem can be eliminated, e.g., by removalof attachment groups located at the functional site or by blocking thefunctional site prior to conjugation. These latter strategies constitutefurther embodiments of the invention (the first strategy beingexemplified further above, e.g. by removal of lysine residues which maybe located close to a functional site). More specifically, according tothe second strategy the conjugation between the interferon β polypeptideand the non-polypeptide moiety is conducted under conditions where thefunctional site of the polypeptide is blocked by a helper moleculecapable of binding to the functional site of the polypeptide.Preferably, the helper molecule is one, which specifically recognizes afunctional site of the polypeptide, such as a receptor, in particularthe type I interferon receptor. Alternatively, the helper molecule maybe an antibody, in particular a monoclonal antibody recognizing theinterferon β polypeptide. In particular, the helper molecule may be aneutralizing monoclonal antibody.

[0230] The polypeptide is allowed to interact with the helper moleculebefore effecting conjugation. This ensures that the functional site ofthe polypeptide is shielded or protected and consequently unavailablefor derivatization by the non-polypeptide moiety such, as a polymer.Following its elution from the helper molecule, the conjugate betweenthe non-polypeptide moiety and the polypeptide can be recovered with atleast a partially preserved functional site.

[0231] The subsequent conjugation of the polypeptide having a blockedfunctional site to a polymer, a lipophilic compound, an organicderivatizing agent or any other compound is conducted in the normal way,e.g. as described in the sections above entitled “Conjugation to . . .”.

[0232] Irrespective of the nature of the helper molecule to be used toshield the functional site of the polypeptide from conjugation, it isdesirable that the helper molecule is free from or comprises only a fewattachment groups for the non-polypeptide moiety of choice in part(s) ofthe molecule, where the conjugation to such groups will hamper thedesorption of the conjugated polypeptide from the helper molecule.Hereby, selective conjugation to attachment groups present innon-shielded parts of the polypeptide can be obtained and it is possibleto reuse the helper molecule for repeated cycles of conjugation. Forinstance, if the non-polypeptide moiety is a polymer molecule such asPEG, which has the epsilon amino group of a lysine or N-terminal aminoacid residue as an attachment group, it is desirable that the helpermolecule is substantially free from conjugatable epsilon amino groups,preferably free from any epsilon amino groups. Accordingly, in apreferred embodiment the helper molecule is a protein or peptide capableof binding to the functional site of the polypeptide, which protein orpeptide is free from any conjugatable attachment groups for thenon-polypeptide moiety of choice.

[0233] In a further embodiment, the helper molecule is first covalentlylinked to a solid phase such as column packing materials, for instanceSephadex or agarose beads, or a surface, e.g. reaction vessel.Subsequently, the polypeptide is loaded onto the column materialcarrying the helper molecule and conjugation carried out according tomethods known in the art, e.g. as described in the sections aboveentitled “Conjugation to . . . ”. This procedure allows the polypeptideconjugate to be separated from the helper molecule by elution. Thepolypeptide conjugate is eluated by conventional techniques underphysico-chemical conditions that do not lead to a substantivedegradation of the polypeptide conjugate. The fluid phase containing thepolypeptide conjugate is separated from the solid phase to which thehelper molecule remains covalently linked. The separation can beachieved in other ways: For instance, the helper molecule may bederivatised with a second molecule (e.g. biotin) that can be recognizedby a specific binder (e.g. streptavidin). The specific binder may belinked to a solid phase thereby allowing the separation of thepolypeptide conjugate from the helper molecule-second molecule complexthrough passage over a second helper-solid phase column which willretain, upon subsequent elution, the helper molecule-second moleculecomplex, but not the polypeptide conjugate. The polypeptide conjugatemay be released from the helper molecule in any appropriate fashion.De-protection may be achieved by providing conditions in which thehelper molecule dissociates from the functional site of the interferon βto which it is bound. For instance, a complex between an antibody towhich a polymer is conjugated and an anti-idiotypic antibody can bedissociated by adjusting the pH to an acid or alkaline pH.

[0234] Conjugation of a Tagged Interferon β Polypeptide

[0235] In an alternative embodiment the interferon β polypeptide isexpressed, as a fusion protein, with a tag, i.e. an amino acid sequenceor peptide stretch made up of typically 1-30, such as 1-20 or 1-15 or1-10 amino acid residues. Besides allowing for fast and easypurification, the tag is a convenient tool for achieving conjugationbetween the tagged polypeptide and the non-polypeptide moiety. Inparticular, the tag may be used for achieving conjugation in microtiterplates or other carriers, such as paramagnetic beads, to which thetagged polypeptide can be immobilised via the tag. The conjugation tothe tagged polypeptide in, e.g., microtiter plates has the advantagethat the tagged polypeptide can be immobilised in the microtiter platesdirectly from the culture broth (in principle without any purification)and subjected to conjugation. Thereby, the total number of process steps(from expression to conjugation) can be reduced. Furthermore, the tagmay function as a spacer molecule ensuring an improved accessibility tothe immobilised polypeptide to be conjugated. The conjugation using atagged polypeptide may be to any of the non-polypeptide moietiesdisclosed herein, e.g. to a polymer molecule such as PEG.

[0236] The identity of the specific tag to be used is not critical aslong as the tag is capable of being expressed with the polypeptide andis capable of being immobilised on a suitable surface or carriermaterial. A number of suitable tags are commercially available, e.g.from Unizyme Laboratories, Denmark. For instance, the tag may be any ofthe following sequences:

[0237] His-His-His-His-His-His

[0238] Met-Lys-His-His-His-His-His-His

[0239] Met-Lys-His-His-Ala-His-His-Gln-His-His

[0240] Met-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln

[0241] (vectors useful for providing such tags are available fromUnizyme Laboratories, Denmark)

[0242] or any of the following:

[0243] EQKLI SEEDL (a C-terminal tag described in Mol. Cell. Biol.5:3610-16, 1985)

[0244] DYKDDDDK (a C- or N-terminal tag)

[0245] YPYDVPDYA

[0246] Antibodies against the above tags are commercially available,e.g. from ADI, Aves Lab and Research Diagnostics.

[0247] A convenient method for using a tagged polypeptide for PEGylationis given in the Materials and Methods section below.

[0248] The subsequent cleavage of the tag from the polypeptide may beachieved by use of commercially available enzymes.

[0249] Polypeptides of the Invention

[0250] In further aspects the invention relates to generally novelinterferon β polypeptides described herein that, as compared to humanwildtype interferon β has at least one introduced and/or at least oneremoved attachment group for a non-polypeptide moiety. The novelpolypeptides are important intermediate compounds for the preparation ofa conjugate of the invention. In addition, the polypeptides themselvesmay have interesting properties.

[0251] Examples of such polypeptides include those that comprises anamino acid sequence which differs from that of wild-type humaninterferon β in that at least one amino acid residue selected from thegroup consisting of N4, F8, L9, Q10, R11, S13, L24, N25, G26, L28, E29,N37, F38, Q48, Q49, Q64, N65, I66, F67, A68, I69, F70, R71, Q72, D73,S74, S75, S76, T77, G78, W79, N80, E81, T82, I83, V84, L87, L88, A89,N90, V91, T92, H93, Q94, D110, F111, T112, R113, R128, H140, T144, I145,R147, V148, L151, R152, F154, Y155, N158 and N166 is replaced with adifferent amino acid residue selected from the group consisting of K, R,D, E, C and N. The amino acid residues specified above are located inpositions, which are exposed at the surface of human interferon βmolecule as demonstrated by the solved 3D structure of human interferonβ. By replacing one or more of these residue with either of K, R, D, E,C and N attachment group(s) for a non-polypeptide moiety, in particulara polymer attachment group or an amino acid residue susceptible tomodification by a carbohydrate moiety, is/are introduced into humaninterferon β. The resulting modified human interferon β molecule is asuitable starting compound for the preparation of an interferon βconjugate having improved properties as compared to the unmodified humaninterferon β molecule.

[0252] In a further aspect the invention relates to an interferon βpolypeptide comprising an amino acid sequence which differs from that ofwild-type human interferon β in that at least one amino acid residueselected from the group consisting of N4, F8, L9, Q10, R11, S12, S13,L24, N25, G26, L28, E29, N37, F38, D39, Q48, Q49, Q64, N65, I66, F67,A68, I69, F70, R71, Q72, D73, S74, S75, S76, T77, G78, W79, N80, E81,T82, I83, V84, E85, L87, L88, A89, N90, V91, Y92, H93, Q94, D110, F111,T112, R113, R128, H140, T144, I145, R147, V148, L151, R152, F154, Y155,N158, G162, and N166 is replaced with a lysine residue, provided thatthe polypeptide is different from the one having the amino acid sequenceof wild-type human interferon β with the following substitutions:D54N+E85K+V911+V101M and different from one which is a hybrid moleculebetween interferon β and interferon α which as a consequence of being ahybrid has a lysine in position 39. The first of the disclaimedpolypeptides is disclosed by Stewart et al, DNA Vol 6 no2 1987 p119-128and was found to be inactive, the second is disclosed in U.S. Pat. No.4,769,233 and was constructed with the purpose of improving thebiological activity of interferon β. None of the disclaimed polypeptideswere made for or described as being suitable intermediates for thepreparation of interferon β conjugates with reduced immunogenicityand/or prolonged functional in vivo half-life and/or serum half-life.

[0253] A still further example includes an interferon β polypeptidecomprising an amino acid sequence which differs from that of SEQ ID NO 2in one or more substitutions selected from the group consisting of N4K,F15K, Q16K, R27K, R35K, D39K, Q49K, E85K, A89K, E103K, E109K, R124K,E137K and R159K, provided that when the substitution is R27K thepolypeptide is different from the one having the amino acid sequence ofwild-type human interferon β with the following substitutions:R27K+E43K. The disclaimed polypeptide is disclosed by Stewart et al, DNAVol 6 no2 1987 p119-128 and was found to have a low activity. Thepolypeptide was made in the course of a study of function-structurerelationship and was not mentioned as a possible intermediate productfor the preparation of improved interferon β conjugate molecules. Forinstance, the interferon β polypeptide comprises an amino acid sequence,which differs from that of SEQ ID NO 2 in that it comprises thesubstitution R27K in combination with at least one additionalsubstitution that is different from E43K, or the substitution R35K incombination with at least one additional substitution provided that thepolypeptide has an amino acid sequence which is different from the aminoacid sequence of wild-type human interferon β modified with thefollowing substitutions: G7E+S12N+C17Y+R35K. The disclaimed polypeptideis disclosed by Stewart et al, DNA Vol 6 no2 1987 p119-128 as having aretained antiproliferative activity on Daudi cells relative to theirantiviral activity, but reduced overall activity as compared to wildtype interferon β. The disclaimed polypeptide was not prepared with thepurpose of reducing the immunogenicity and/or increasing the functionalin vivo half-life and/or serum half-life, but was made in the course ofa study of the structural functional relationship of interferon β.

[0254] The polypeptide of the invention may, in addition to any of theabove specified substitutions, additionally comprise the substitutionC17S and/or a deletion of M1 or the substitution M1K. Furthermore, thepolypeptide of the invention may comprise an amino acid sequence, whichfurther differs from that of SEQ ID NO 2 in the removal, preferably bysubstitution, of at least one lysine residue selected from the groupconsisting of K19, K33, K45, K52, K99, K105, K108, K115, K123, K134, andK136. The lysine residue(s) may be replaced with any other amino acidresidue, but is preferably replaced by an arginine or a glutamine. Inparticular, the polypeptide of the invention may be one, wherein K45,K52 and/or K123 has/have been replaced with another amino acid residue,but preferably an arginine or a glutamine residue. Also, the polypeptidemay be expressed with a tag, e.g. as described in the section furtherabove entitled “Conjugation of a tagged interferon β polypeptide”.

[0255] A still further example of an interferon β polypeptide of theinvention includes one, that comprises an amino acid sequence whichdiffers from that of wild-type human interferon β in that at least onelysine residue selected from the group consisting of K19, K33, K45, K52,K99, K105, K108, K115, K123, K134, and K136 has been replaced with anyother amino acid residue, provided that the interferon β polypeptide isdifferent from a hybrid between interferon β and interferon α, which asa consequence of being a hybrid has a phenylalanine in position 45.Preferably, at least K19, K45, K52 and/or K123 is/are are replaced.While the lysine residue may be deleted in accordance with this aspectof the invention, it is preferred that it be replaced with any otheramino acid residue, preferably an arginine or a glutamine. Normally, thepolypeptide of the invention comprises an amino acid sequence whichdiffers in 1-15 amino acid residues from the amino acid sequence shownin SEQ ID NO 2 as further discussed above. Examples of polypeptides ofthe invention are selected from the group consisting of those thatcomprises an amino acid sequence, which differs from that of SEQ ID NO 2in at least the following substitutions:

[0256] R27K+R159K;

[0257] R27K+K45R+R159K;

[0258] R27K+Q49K+E85K+A89K;

[0259] R27K+K45R+Q49K+E85K+A89K;

[0260] R27K+D39K+Q49K+E85K+A89K;

[0261] R27K+D39K+K45R+Q49K+E85K+A89K;

[0262] N4K+R27K+D39K+Q49K+E85K+A89K;

[0263] N4K+R27K+D39K+K45R+Q49K+E85K+A89K;

[0264] R27K+K123R+R159K;

[0265] R27K+K45R+K123R+R159K;

[0266] R27K+Q49K+E85K+A89K+K123R;

[0267] R27K+K45R+Q49K+E85K+A89K+K123R;

[0268] R27K+D39K+Q49K+E85K+A89K+K123R;

[0269] R27K+D39K+K45R+Q49K+E85K+A89K+K123R;

[0270] N4K+R27K+D39K+Q49K+E85K+A89K+K123R; and

[0271] N4K+R27K+D39K+K45R+Q49K+E85K+A89K+K123R.

[0272] It will be understood that any of the polypeptides of theinvention disclosed herein may be used to prepare a conjugate of theinvention, i.e. be covalently coupled to any of the non-polypeptidemoieties disclosed herein. In particular, when a polypeptide of theinvention is expressed in a glycosylating microorganism the polypeptidemay be provided in glycosylated form.

[0273] Methods of Preparing an Interferon β Polypeptide for Use in theInvention

[0274] The polypeptide of the present invention or the polypeptide partof a conjugate of the invention, optionally in glycosylated form, may beproduced by any suitable method known in the art. Such methods includeconstructing a nucleotide sequence encoding the polypeptide andexpressing the sequence in a suitable transformed or transfected host.However, polypeptides of the invention may be produced, albeit lessefficiently, by chemical synthesis or a combination of chemicalsynthesis or a combination of chemical synthesis and recombinant DNAtechnology.

[0275] The nucleotide sequence of the invention encoding an interferon βpolypeptide may be constructed by isolating or synthesizing a nucleotidesequence encoding the parent interferon β, e.g. with the amino acidsequence shown in SEQ ID NO 2, and then changing the nucleotide sequenceso as to effect introduction (i.e. insertion or substitution) ordeletion (i.e. removal or substitution) of the relevant amino acidresidue(s).

[0276] The nucleotide sequence is conveniently modified by site-directedmutagenesis in accordance with well-known methods, see, e.g., Mark etal., “Site-specific Mutagenesis of the Human Fibroblast InterferonGene”, Proc. Natl. Acad. Sci. USA, 81, pp. 5662-66 (1984); and U.S. Pat.No. 4,588,585.

[0277] Alternatively, the nucleotide sequence is prepared by chemicalsynthesis, e.g. by using an oligonucleotide synthesizer, whereinoligonucleotides are designed based on the amino acid sequence of thedesired polypeptide, and preferably selecting those codons that arefavored in the host cell in which the recombinant polypeptide will beproduced. For example, several small oligonucleotides coding forportions of the desired polypeptide may be synthesized and assembled byPCR, ligation or ligation chain reaction (LCR). The individualoligonucleotides typically contain 5′ or 3′ overhangs for complementaryassembly.

[0278] Once assembled (by synthesis, site-directed mutagenesis oranother method), the nucleotide sequence encoding the interferon βpolypeptide is inserted into a recombinant vector and operably linked tocontrol sequences necessary for expression of the interferon β in thedesired transformed host cell.

[0279] It should of course be understood that not all vectors andexpression control sequences function equally well to express thenucleotide sequence encoding a polypeptide variant described herein.Neither will all hosts function equally well with the same expressionsystem. However, one of skill in the art may make a selection amongthese vectors, expression control sequences and hosts without undueexperimentation. For example, in selecting a vector, the host must beconsidered because the vector must replicate in it or be able tointegrate into the chromosome. The vector's copy number, the ability tocontrol that copy number, and the expression of any other proteinsencoded by the vector, such as antibiotic markers, should also beconsidered. In selecting an expression control sequence, a variety offactors should also be considered. These include, for example, therelative strength of the sequence, its controllability, and itscompatibility with the nucleotide sequence encoding the polypeptide,particularly as regards potential secondary structures. Hosts should beselected by consideration of their compatibility with the chosen vector,the toxicity of the product coded for by the nucleotide sequence, theirsecretion characteristics, their ability to fold the polypeptidecorrectly, their fermentation or culture requirements, and the ease ofpurification of the products coded for by the nucleotide sequence.

[0280] The recombinant vector may be an autonomously replicating vector,i.e. a vector which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g. aplasmid. Alternatively, the vector is one which, when introduced into ahost cell, is integrated into the host cell genome and replicatedtogether with the chromosome(s) into which it has been integrated.

[0281] The vector is preferably an expression vector, in which thenucleotide sequence encoding the polypeptide of the invention isoperably linked to additional segments required for transcription of thenucleotide sequence. The vector is typically derived from plasmid orviral DNA. A number of suitable expression vectors for expression in thehost cells mentioned herein are commercially available or described inthe literature. Useful expression vectors for eukaryotic hosts, include,for example, vectors comprising expression control sequences from SV40,bovine papilloma virus, adenovirus and cytomegalovirus. Specific vectorsare, e.g., pCDNA3.1(+)\Hyg (Invitrogen, Carlsbad, Calif., USA) andpCI-neo (Stratagene, La Jola, Calif., USA). Useful expression vectorsfor bacterial hosts include known bacterial plasmids, such as plasmidsfrom E. coli, including pBR322, pET3a and pET12a (both from NovagenInc., WI., USA), wider host range plasmids, such as RP4, phage DNAs,e.g., the numerous derivatives of phage lambda, e.g., NM989, and otherDNA phages, such as M13 and filamentous single stranded DNA phages.Useful expression vectors for yeast cells include the 2μ plasmid andderivatives thereof, the POT1 vector (U.S. Pat. No. 4,931,373), thepJSO37 vector described in (Okkels,. Ann. New York Acad. Sci. 782,202-207, 1996) and pPICZ A, B or C (Invitrogen). Useful vectors forinsect cells include pVL941, pBG311 (Cate et al., “Isolation of theBovine and Human Genes for Mullerian Inhibiting Substance And Expressionof the Human Gene In Animal Cells”, Cell, 45, pp. 685-98 (1986),pBluebac 4.5 and pMelbac (both available from Invitrogen).

[0282] Other vectors for use in this invention include those that allowthe nucleotide sequence encoding the polypeptide variant to be amplifiedin copy number. Such amplifiable vectors are well known in the art. Theyinclude, for example, vectors able to be amplified by DHFR amplification(see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufinan and Sharp,“Construction Of A Modular Dihydrofolate Reductase cDNA Gene: AnalysisOf Signals Utilized For Efficient Expression”, Mol. Cell. Biol., 2, pp.1304-19 (1982)) and glutamine synthetase (“GS”) amplification (see,e.g., U.S. Pat. No. 5,122,464 and EP 338,841).

[0283] The recombinant vector may further comprise a DNA sequenceenabling the vector to replicate in the host cell in question. Anexample of such a sequence (when the host cell is a mammalian cell) isthe SV40 origin of replication. When the host cell is a yeast cell,suitable sequences enabling the vector to replicate are the yeastplasmid 2μ replication genes REP 1-3 and origin of replication.

[0284] The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the genecoding for dihydrofolate reductase (DHFR) or the Schizosaccharomycespombe TPI gene (described by P. R. Russell, Gene 40, 1985, pp. 125-130),or one which confers resistance to a drug, e.g. ampicillin, kanamycin,tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate. Forfilamentous fungi, selectable markers include amdS, pyrG, arcB, niaD,sC.

[0285] The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression ofthe polypeptide of the invention. Each control sequence may be native orforeign to the nucleic acid sequence encoding the polypeptide. Suchcontrol sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, enhancer orupstream activating sequence, signal peptide sequence, and transcriptionterminator. At a minimum, the control sequences include a promoter.

[0286] A wide variety of expression control sequences may be used in thepresent invention. Such useful expression control sequences include theexpression control sequences associated with structural genes of theforegoing expression vectors as well as any sequence known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof.

[0287] Examples of suitable control sequences for directingtranscription in mammalian cells include the early and late promoters ofSV40 and aderiovirus, e.g. the adenovirus 2 major late promoter, theMT-1 (metallothionein gene) promoter, the human cytomegalovirusimmediate-early gene promoter (CMV), the human elongation factor 1α(EF-1α) promoter, the Drosophila minimal heat shock protein 70 promoter,the Rous Sarcoma Virus (RSV) promoter, the human ubiquitin C (UbC)promoter, the human growth hormone terminator, SV40 or adenovirus Elbregion polyadenylation signals and the Kozak consensus sequence (Kozak,M. J Mol Biol Aug. 20, 1987;196(4):947-50).

[0288] In order to improve expression in mammalian cells a syntheticintron may be inserted in the 5′ untranslated region of the nucleotidesequence encoding the polypeptide of interest. An example of a syntheticintron is the synthetic intron from the plasmid pCI-Neo (available fromPromega Corporation, Wis., USA).

[0289] Examples of suitable control sequences for directingtranscription in insect cells include the polyhedrin promoter, the P10promoter, the Autographa californica polyhedrosis virus basic proteinpromoter, the baculovirus immediate early gene 1 promoter and thebaculovirus 39K delayed-early gene promoter, and the SV40polyadenylation sequence.

[0290] Examples of suitable control sequences for use in yeast hostcells include the promoters of the yeast α-mating system, the yeasttriose phosphate isomerase (TPI) promoter, promoters from yeastglycolytic genes or alcohol dehydogenase genes, the ADH2-4c promoter andthe inducible GAL promoter.

[0291] Examples of suitable control sequences for use in filamentousfungal host cells include the ADH3 promoter and terminator, a promoterderived from the genes encoding Aspergillus oryzae TAKA amylase triosephosphate isomerase or alkaline protease, an A. niger α-amylase, A.niger or A. nidulans glucoamylase, A. nidulans acetamidase, Rhizomucormiehei aspartic proteinase or lipase, the TPI1 terminator and the ADH3terminator.

[0292] Examples of suitable control sequences for use in bacterial hostcells include promoters of the lac system, the trp system, the TAC orTRC system and the major promoter regions of phage lambda.

[0293] The nucleotide sequence of the invention encoding an interferon βpolypeptide, whether prepared by site-directed mutagenesis, synthesis orother methods, may or may not also include a nucleotide sequence thatencode a signal peptide. The signal peptide is present when thepolypeptide is to be secreted from the cells in which it is expressed.Such signal peptide, if present, should be one recognized by the cellchosen for expression of the polypeptide. The signal peptide may behomologous (e.g. be that normally associated with human interferon β) orheterologous (i.e. originating from another source than human interferonβ) to the polypeptide or may be homologous or heterologous to the hostcell, i.e. be a signal peptide normally expressed from the host cell orone which is not normally expressed from the host cell. Accordingly, thesignal peptide may be prokaryotic, e.g. derived from a bacterium such asE. coli, or eukaryotic, e.g. derived from a mammalian, or insect oryeast cell.

[0294] The presence or absence of a signal peptide will, e.g., depend onthe expression host cell used for the production of the polypeptide, theprotein to be expressed (whether it is an intracellular or extracellularprotein) and whether it is desirable to obtain secretion. For use infilamentous fungi, the signal peptide may conveniently be derived from agene encoding an Aspergillus sp. amylase or glucoamylase, a geneencoding a Rhizomucor miehei lipase or protease or a Humicola lanuginosalipase. The signal peptide is preferably derived from a gene encoding A.oryzae TAKA amylase, A. niger neutral α-amylase, A. niger acid-stableamylase, or A. niger glucoamylase. For use in insect cells, the signalpeptide may conveniently be derived from an insect gene (cf. WO90/05783), such as the lepidopteran Manduca sexta adipokinetic hormoneprecursor, (cf. U.S. Pat. No. 5,023,328), the honeybee melittin(Invitrogen), ecdysteroid UDPglucosyltransferase (egt) (Murphy et al.,Protein Expression and Purification 4, 349-357 (1993) or humanpancreatic lipase (hpl) (Methods in Enzymology 284, pp. 262-272, 1997).A preferred signal peptide for use in mammalian cells is that of humaninterferon β apparent from the examples hereinafter or the murine Igkappa light chain signal peptide (Coloma, M (1992) J. Imm. Methods152:89-104). For use in yeast cells suitable signal peptides have beenfound to be the α-factor signal peptide from S. cereviciae. (cf. U.S.Pat. No. 4,870,008), the signal peptide of mouse salivary amylase (cf.O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), a modifiedcarboxypeptidase signal peptide (cf. L. A. Valls et al., Cell 48, 1987,pp. 887-897), the yeast BAR1 signal peptide (cf. WO 87/02670), and theyeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani etal., Yeast 6, 1990, pp. 127-137).

[0295] Any suitable host may be used to produce the interferon βpolypeptide, including bacteria, fungi (including yeasts), plant,insect, mammal, or other appropriate animal cells or cell lines, as wellas transgenic animals or plants. Examples of bacterial host cellsinclude grampositive bacteria such as strains of Bacillus, e.g. B.brevis or B. subtilis, Pseudomonas or Streptomyces, or gramnegativebacteria, such as strains of E. coli. The introduction of a vector intoa bacterial host cell may, for instance, be effected by protoplasttransformation (see, e.g., Chang and Cohen, 1979, Molecular GeneralGenetics 168: 111-115), using competent cells (see, e.g., Young andSpizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221),electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, Journalof Bacteriology 169: 5771-5278).

[0296] Examples of suitable filamentous fungal host cells includestrains of Aspergillus, e.g. A. oryzae, A. niger, or A. nidulans,Fusarium or Trichoderma. Fungal cells may be transformed by a processinvolving protoplast formation, transformation of the protoplasts, andregeneration of the cell wall in a manner known per se. Suitableprocedures for transformation of Aspergillus host cells are described inEP 238 023 and U.S. Pat. No. 5,679,543. Suitable methods fortransforming Fusarium species are described by Malardier et al., 1989,Gene 78: 147-156 and WO 96/00787. Yeast may be transformed using theprocedures described by Becker and Guarente, In Abelson, J. N. andSimon, M. I., editors, Guide to Yeast Genetics and Molecular Biology,Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., NewYork; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen etal., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

[0297] Examples of suitable yeast host cells include strainsof.Saccharomyces, e.g. S. cerevisiae, Schizosaccharomyces, Klyveromyces,Pichia, such as P. pastoris or P. methanolica, Hansenula, such as H.Polymorpha or Yarrowia. Methods for transforming yeast cells withheterologous DNA and producing heterologous polypeptides therefrom aredisclosed by Clontech Laboratories, Inc, Palo Alto, Calif., USA (in theproduct protocol for the Yeastmaker™ Yeast Tranformation System Kit),and by Reeves et al., FEMS Microbiology Letters 99 (1992) 193-198,Manivasakam and Schiestl, Nucleic Acids Research, 1993, Vol. 21, No. 18,pp. 4414-4415 and Ganeva et al., FEMS Microbiology Letters 121 (1994)159-164.

[0298] Examples of suitable insect host cells include a Lepidoptora cellline, such as Spodoptera frugiperda (Sf9 or Sf21) or Trichoplusioa nicells (High Five) (U.S. Pat. No. 5,077,214). Transformation of insectcells and production of heterologous polypeptides therein may beperformed as described by Invitrogen.

[0299] Examples of suitable mammalian host cells include Chinese hamsterovary (CHO) cell lines, (e.g. CHO-K1; ATCC CCL-61), Green Monkey celllines (COS) (e.g. COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mousecells (e.g. NS/O), Baby Hamster Kidney (BHK) cell lines (e.g. ATCCCRL-1632 or ATCC CCL-10), and human cells (e.g. HEK 293 (ATCCCRL-1573)), as well as plant cells in tissue culture. Additionalsuitable cell lines are known in the art and available from publicdepositories such as the American Type Culture Collection, Rockville,Md. Also, the mammalian cell, such as a CHO cell, may be modified toexpress sialyltransferase, e.g. 1,6-sialyltransferase, e.g. as describedin U.S. Pat. No. 5,047,335, in order to provide improved glycosylationof the interferon β polypeptide.

[0300] Methods for introducing exogeneous DNA into mammalian host cellsinclude calcium phosphate-mediated transfection, electroporation,DEAE-dextran mediated transfection, liposome-mediated transfection,viral vectors and the transfection methods described by LifeTechnologies Ltd, Paisley, UK using Lipofectamin 2000 and RocheDiagnostics Corporation, Indianapolis, USA using FuGENE 6. These methodsare well known in the art and e.g. described by Ausbel et al. (eds.),1996, Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, USA. The cultivation of mammalian cells are conducted according toestablished methods, e.g. as disclosed in (Animal Cell Biotechnology,Methods and Protocols, Edited by Nigel Jenkins, 1999, Human Press Inc,Totowa, N.J., USA and Harrison M A and Rae I F, General Techniques ofCell Culture, Cambridge University Press 1997).

[0301] In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermenters performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

[0302] The resulting polypeptide may be recovered by methods known inthe art. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray drying, evaporation, orprecipitation.

[0303] The polypeptides may be purified by a variety of procedures knownin the art including, but not limited to, chromatography (e.g., ionexchange, affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989). Specificmethods for purifying polypeptides exhibiting interferon β activity aredisclosed in U.S. Pat. Nos. 4,289,689, 4,359,389, 4,172,071, 4,551,271,5,244,655, 4,485,017, 4,257,938 and 4,541,952. A specific purificationmethod is based on immunoaffinity purification (see, e.g., Okamura etal., “Human Fibroblastoid Interferon: Immunosorbent ColumnChromatography And N-Terminal Amino Acid Sequence”, Biochem., 19, pp.3831-35 (1980)). Furthermore, purification may be based on the use ofIFNAR 1 and/or IFNAR 2, in particular IFNAR 2.

[0304] The biological activity of the interferon β polypeptides can beassayed by any suitable method known in the art. Such assays includeantibody neutralization of antiviral activity, induction of proteinkinase, oligoadenylate 2,5-A synthetase or phosphodiesterase activities,as described in EP 41313 B 1. Such assays also include immunomodulatoryassays (see, e.g., U.S. Pat. No. 4,753,795), growth inhibition assays,and measurement of binding to cells that express interferon receptors.Specific assays for determining the biological activity of polypeptidesor conjugates of the invention are disclosed in the Materials andMethods section hereinafter.

[0305] Cell Culture of the Invention

[0306] In a further aspect the invention relates to a cell culturecomprising a) a host cell transformed with a nucleotide sequenceencoding a polypeptide exhibiting interferon β activity, and b) aculture medium comprising said polypeptide produced by expression ofsaid nucleotide sequence in a concentration of at least 800,000 IU/ml ofmedium, preferably in a concentration in the range of 800,000-3,500,000IU/ml medium. While the polypeptide exhibiting interferon α activity maybe a wild-type interferon β, e.g. human interferon β or a variantthereof (e.g. interferon β 1a or 1b) the polypeptide is preferably aninterferon β polypeptide as described herein:

[0307] In a still further aspect the invention relates to a method ofproducing an interferon β polypeptide as described herein, the methodcomprising:

[0308] (a) culturing a cell expressing an interferon β polypeptidevariant in a culture medium, such that the concentration of theinterferon β polypeptide variant in the medium is at least 800,000 IU/mlmedium, in particular in the range of between 800,000 and 3,500,000IU/ml medium; and

[0309] (b) recovering the interferon β polypeptide.

[0310] Other Methods of the Invention

[0311] In a still further aspect the invention relates to a methodreducing immunogenicity and/or of increasing functional in vivohalf-life and/or serum half-life of an interferon β polypeptide, whichmethod comprises introducing an amino acid residue constituting anattachment group for a first non-polypeptide moiety into a positionexposed at the surface of the protein that does not contain such groupand/or removing an amino acid residue constituting an attachment groupfor a first non-polypeptide moiety and subjecting the resulting modifiedpolypeptide to conjugation with the first non-polypeptide moiety.

[0312] Preferably, the amino acid residue to be introduced and/orremoved is as defined in the present application. The non-polypeptidemoiety is normally selected from the group consisting of a polymermolecule, a sugar moiety, a lipophilic group and an organic derivatizingagent.

[0313] In a still further aspect the invention relates to a method forpreparing a conjugate of the invention, wherein the interferon βpolypeptide is reacted with the non-polypeptide moiety to which it is tobe conjugated under conditions conducive for the conjugation to takeplace, and the conjugate is recovered.

[0314] Pharmaceutical Compositition and Uses of a Conjugate of theInvention

[0315] The interferon β polypeptide or the conjugate of the invention isadministered at a dose approximately paralleling that employed intherapy with human interferon β such as Avonex™, Rebif® and Betaseron®,or a higher dosis. The exact dose to be administered depends on thecircumstances. Normally, the dose should be capable of preventing orlessening the severity or spread of the condition or indication beingtreated. It will be apparent to those of skill in the art that aneffective amount of a polypeptide, conjugate or composition of theinvention depends, inter alia, upon the disease, the dose, theadministration schedule, whether the polypeptide or conjugate orcomposition is administered alone or in conjunction with othertherapeutic agents, the serum half-life of the compositions, and thegeneral health of the patient.

[0316] The polypeptide or conjugate of the invention can be used “as is”and/or in a salt form thereof. Suitable salts include, but are notlimited to, salts with alkali metals or alkaline earth metals, such assodium, potassium, lithium, calcium and magnesium, as well as e.g. zincsalts. These salts or complexes may by present as a crystalline and/oramorphous structure.

[0317] The polypeptide or conjugate of the invention is preferablyadministered in a composition including a pharmaceutically acceptablecarrier or excipient. “Pharmaceutically acceptable” means a carrier orexcipient that does not cause any untoward effects in patients to whomit is administered. Such pharmaceutically acceptable carriers andexcipients are well known in the art.

[0318] The polypeptide or conjugate of the invention can be formulatedinto pharmaceutical compositions by well-known methods. Suitableformulations are described in U.S. Pat. No. 5,183,746, Remington'sPharmaceutical Sciences by E. W.Martin, 18^(th) edition, A. R. Gennaro,Ed., Mack Publishing Company [990]; Pharmaceutical FormulationDevelopment of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds.,Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rdedition, A. Kibbe, Ed., Pharmaceutical Press [2000]).

[0319] The pharmaceutical composition of the polypeptide or conjugate ofthe invention may be formulated in a variety of forms, including liquid,gel, lyophilized, pulmonary dispersion, or any other suitable form, e.g.as a compressed solid. The preferred form will depend upon theparticular indication being treated and will be apparent to one of skillin the art.

[0320] The pharmaceutical composition containing the polypeptide orconjugate of the invention may be administered orally, intravenously,intracerebrally, intramuscularly, intraperitoneally, intradermally,subcutaneously, intranasally, intrapulmonary, by inhalation, or in anyother acceptable manner, e.g. using PowderJect or ProLease technology.The preferred mode of administration will depend upon the particularindication being treated and will be apparent to one of skill in theart.

[0321] Parenterals

[0322] An example of a pharmaceutical composition is a solution designedfor parenteral administration. Although in many cases pharmaceuticalsolution formulations are provided in liquid form, appropriate forimmediate use, such parenteral formulations may also be provided infrozen or in lyophilized form. In the former case, the composition mustbe thawed prior to use. The latter form is often used to enhance thestability of the active compound contained in the composition under awider variety of storage conditions, as it is recognized by thoseskilled in the art that lyophilized preparations are generally morestable than their liquid counterparts. Such lyophilized preparations arereconstituted prior to use by the addition of one or more suitablepharmaceutically acceptable diluents such as sterile water for injectionor sterile physiological saline solution.

[0323] In case of parenterals, they are prepared for storage aslyophilized formulations or aqueous solutions by mixing, as appropriate,the polypeptide having the desired degree of purity with one or morepharmaceutically acceptable carriers, excipients or stabilizerstypically employed in the art (all of which are termed “excipients”),for example buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants and/or othermiscellaneous additives.

[0324] Buffering agents help to maintain the pH in the range whichapproximates physiological conditions. They are typically present at aconcentration ranging from about 2 mM to about 50 mM Suitable bufferingagents for use with the present invention include both organic andinorganic acids and salts thereof such as citrate buffers (e.g.,monosodium citrate-disodium citrate mixture, citric acid-trisodiumcitrate mixture, citric acid-monosodium citrate mixture, etc.),succinate buffers (e.g., succinic acid-monosodium succinate mixture,succinic acid-sodium hydroxide mixture, succinic acid-disodium succinatemixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartratemixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodiumhydroxide mixture, etc.), fumarate buffers (e.g., fimaricacid-monosodium fimarate mixture, fumaric acid-disodium fumaratemixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconatebuffers (e.g., gluconic acid-sodium glyconate mixture, gluconicacid-sodium hydroxide mixture, gluconic acid-potassium glyuconatemixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalatemixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassiumoxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodiumlactate mixture, lactic acid-sodium hydroxide mixture, lacticacid-potassium lactate mixture, etc.) and acetate buffers (e.g., aceticacid-sodium acetate mixture, acetic acid-sodium hydroxide mixture,etc.). Additional possibilities are phosphate buffers, histidine buffersand trimethylamine salts such as Tris.

[0325] Preservatives are added to retard microbial growth, and aretypically added in amounts of about 0.2%-1% (w/v). Suitablepreservatives for use with the present invention include phenol, benzylalcohol, meta-cresol, methyl paraben, propyl paraben,octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g.benzalkonium chloride, bromide or iodide), hexamethonium chloride, alkylparabens such as methyl or propyl paraben, catechol, resorcinol,cyclohexanol and 3-pentanol.

[0326] Isotonicifiers are added to ensure isotonicity of liquidcompositions and include polyhydric sugar alcohols, preferably trihydricor higher sugar alcohols, such as glycerin, erythritol, arabitol,xylitol, sorbitol and mannitol. Polyhydric alcohols can be present in anamount between 0.1% and 25% by weight, typically 1% to 5%, taking intoaccount the relative amounts of the other ingredients.

[0327] Stabilizers refer to a broad category of excipients which canrange in function from a bulking agent to an additive which solubilizesthe therapeutic agent or helps to prevent denaturation or adherence tothe container wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, omithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur-containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thiosulfate; low molecular weight polypeptides (i.e. <10residues); proteins such as human serum albumin, bovine serum albumin,gelatin or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructoseand glucose; disaccharides such as lactose, maltose and sucrose;trisaccharides such as raffinose, and polysaccharides such-as dextran.Stabilizers are typically present in the range of from 0.1 to 10,000parts by weight based on the active protein weight.

[0328] Non-ionic surfactants or detergents (also known as “wettingagents”) may be present to help solubilize the therapeutic agent as wellas to protect the therapeutic polypeptide against agitation-inducedaggregation, which also permits the formulation to be exposed to shearsurface stress without causing denaturation of the polypeptide. Suitablenon-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers(184, 188 etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers(Tween®-20, Tween®-80, etc.).

[0329] Additional miscellaneous excipients include bulking agents orfillers (e.g. starch), chelating agents (e.g. EDTA), antioxidants (e.g.,ascorbic acid, methionine, vitamin E) and cosolvents.

[0330] The active ingredient may also be entrapped in microcapsulesprepared, for example, by coascervation techniques or by interfacialpolymerization, for example hydroxymethylcellulose, gelatin orpoly-(methylmethacylate) microcapsules, in colloidal drug deliverysystems (for example liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules) or in macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences, supra.

[0331] Parenteral formulations to be used for in vivo administrationmust be sterile. This is readily accomplished, for example, byfiltration through sterile filtration membranes.

[0332] Sustained Release Preparations

[0333] Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing thepolypeptide or conjugate, the matrices having a suitable form such as afilm or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol)), polylactides, copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the ProLease® technology orLupron Depot® (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid. While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for long periods such asup to or over 100 days, certain hydrogels release proteins for shortertime periods. When encapsulated polypeptides remain in the body for along time, they may denature or aggregate as a result of exposure tomoisture at 37° C., resulting in a loss of biological activity andpossible changes in immunogenicity. Rational strategies can be devisedfor stabilization depending on the mechanism involved. For example, ifthe aggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

[0334] Pulmonary Delivery

[0335] Conjugate formulations suitable for use with a nebulizer, eitherjet or ultrasonic, will typically comprise the conjugate dissolved inwater at a concentration of, e.g., about 0.01 to 25 mg of conjugate permL of solution, preferably about 0.1 to 10 mg/mL. The formulation mayalso include a buffer and a simple sugar (e.g., for proteinstabilization and regulation of osmotic pressure), and/or human serumalbumin ranging in concentration from 0.1 to 10 mg/ml. Examples ofbuffers which may be used are sodium acetate, citrate and glycine.Preferably, the buffer will have a composition and molarity suitable toadjust the solution to a pH in the range of 3 to 9. Generally, buffermolarities of from 1 mM to 50 mM are suitable for this purpose. Examplesof sugars which can be utilized are lactose, maltose, mannitol,sorbitol, trehalose, and xylose, usually in amounts ranging from 1% to10% by weight of the formulation.

[0336] The nebulizer formulation may also contain a surfactant to reduceor prevent surface induced aggregation of the protein caused byatomization of the solution in forming the aerosol. Various conventionalsurfactants can be employed, such as polyoxyethylene fatty acid estersand alcohols, and polyoxyethylene sorbitan fatty acid esters. Amountswill generally range between 0.001% and 4% by weight of the formulation.An especially preferred surfactant for purposes of this invention ispolyoxyethylene sorbitan monooleate.

[0337] Specific formulations and methods of generating suitabledispersions of liquid particles of the invention are described in WO9420069, U.S. Pat. Nos. 5,915,378, 5,960,792, 5,957,124, 5,934,272,5,915,378, 5,855,564, 5,826,570 and 5,522,385 which are herebyincorporated by reference.

[0338] Three specific examples of commercially available nebulizerssuitable for the practice of this invention are the Ultravent nebulizer,manufactured by Mallinckrodt, Inc., St. Louis, Mo., the Acorn IInebulizer, manufactured by Marquest Medical Products, Englewood,Colorado, and the AERx pulmonary drug delivery system manufactured byAradigm Corporation, Hayward, Calif.

[0339] Conjugate formulations for use with a metered dose inhaler devicewill generally comprise a finely divided powder. This powder may beproduced by lyophilizing and then milling a liquid conjugate formulationand may also contain a stabilizer such as human serum albumin (HSA).Typically, more than 0.5% (w/w) HSA is added. Additionally, one or moresugars or sugar alcohols may be added to the preparation if necessary.Examples include lactose maltose, mannitol, sorbitol, sorbitose,trehalose, xylitol, and xylose. The amount added to the formulation canrange from about 0.01 to 200% (w/w), preferably from approximately 1 to50%, of the conjugate present. Such formulations are then lyophilizedand milled to the desired particle size.

[0340] The properly sized particles are then suspended in a propellantwith the aid of a surfactant. The propellant may be any conventionalmaterial employed for this purpose, such as a chlorofluorocarbon, ahydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant. Thismixture is then loaded into the delivery device. An example of acommercially available metered dose inhaler suitable for use in thepresent invention is the Ventolin metered dose inhaler, manufactured byGlaxo Inc., Research Triangle Park, N.C.

[0341] Such conjugate formulations for powder inhalers will comprise afinely divided dry powder containing conjugate and may also include abulking agent, such as lactose, sorbitol, sucrose, or mannitol inamounts which facilitate dispersal of the powder from the device, e.g.,50% to 90% by weight of the formulation. The particles of the powdershall have aerodynamic properties in the lung corresponding to particleswith a density of about 1 g/cm² having a median diameter less than 10micrometers, preferably between 0.5 and 5 micrometers, most preferablyof between 1.5 and 3.5 micrometers.

[0342] An example of a powder inhaler suitable for use in accordancewith the teachings herein is the Spinhaler powder inhaler, manufacturedby Fisons Corp., Bedford, Mass.

[0343] The powders for these devices may be generated and/or deliveredby methods disclosed in U.S. Pat. Nos. 5,997,848, 5,993,783, 5,985,248,5,976,574, 5,922,354, 5,785,049 and 5,5654,007 which are herebyincorporated by reference.

[0344] The pharmaceutical composition containing the conjugate of theinvention may be administered by a wide range of mechanical devicesdesigned for pulmonary delivery of therapeutic products, including butlimited to nebulizers, metered dose inhalers, and powder inhalers, allof which are familiar to those of skill in the art.

[0345] Some specific examples of commercially available devices suitablefor the practice of this invention are the Ultravent nebulizer,manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn IInebulizer, manufactured by Marquest Medical Products, Englewood, Colo.;the Ventolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass.; the “standing cloud” device of InhaleTherapeutic Systems, Inc., San Carlos, Calif.; the AIR inhalermanufactured by Alkermes, Cambridge, Mass.; and the AERx pulmonary drugdelivery system manufactured by Aradigm Corporation, Hayward, Calif.

[0346] The pharmaceutical composition of the invention may beadministered in conjunction with other therapeutic agents. These agentsmay be incorporated as part of the same pharmaceutical composition ormay be administered separately from the polypeptide or conjugate of theinvention, either concurrently or in accordance with any otheracceptable treatment schedule. In addition, the polypeptide, conjugateor pharmaceutical composition of the invention may be used as an adjunctto other therapies.

[0347] Accordingly, this invention provides compositions and methods fortreating most types of viral infections, cancers or tumors (e.g. breastcarcinoma, non-small cell lung cancer) or tumour angiogenesis, Chrohn'sdisease, ulcerative colitis, Guillain-Barrésyndrome, glioma, idiopathicpulmonary fibrosis, abnormal cell growth, or for immunomodulation in anysuitable animal, preferably mammal, and in particular human. Inparticular the polypeptide, conjugate or composition of the inventionmay be used for the treatment of multiple sclerosis (MS), such as any ofthe generally recognized four types of MS (benign, relapsing remittingMS (RRMS), primary progressive MS (PPMS) and secondary progressive MS(SPMS)) and for monosymptomatic MS), hepatitis, or a herpes infection(the latter treatment optionally being combined with a treatment withIL-10).

[0348] In a further aspect the invention relates to a method of treatinga mammal having circulating antibodies against interferon β1a, such asAvonex™ or Rebif®, or 1b, such as Betaseron®, which method comprisesadministering a compound which has the bioactivity of interferon β andwhich has a reduced or no reaction with said antibodies. The compound isadministered in an effective amount. The compound is preferably aconjugate as described herein and the mammal is preferably a humanbeing. The mammals to be treated may suffer from any of the diseaseslisted above for which interferon is a useful treatment. In particular,this aspect of the invention is of interest for the treatment ofmultiple sclerosis (any of the types listed above) or cancer.Furthermore, the invention relates to a method of making apharmaceutical product for use in treatment of mammals havingcirculating antibodies against interferon β 1a, such as Avonex™ orRebif®, or 1b, such as Betaseron®, wherein a compound which has thebioactivity of interferon β and which does not react with such isformulated into an injectable or otherwise suitable formulation. Theterm “circulating antibodies” is intended to indicate antibodies, inparticular neutralizing antibodies, formed in a mammal in response tohaving been treated with any of the commercially available interferon βpreparations (Rebif®, Betaseron®, Avonex™).

[0349] In a further aspect the invention relates to a method of treatinga patient in need of treatment with a pharmaceutical composition with atleast some of the therapeutically beneficial properties of interferon βcomprising administering a composition comprising a compound with atleast part of the therapeutically beneficial activity of interferon β,said treatment having reduced or removed adverse psychological effectsas compared to treatment with interferon β, wherein said compound is anon-naturally occurring conjugate of a polypeptide with interferon βactivity and a non-polypeptide moiety, in particular a conjugateaccording to the present invention.

[0350] In a still further aspect the invention relates to apharmaceutical composition for the treatment of a patient in need oftreatment with a compound having at least part of the therapeuticallybeneficial properties of interferon β, said composition comprising acompound which is a non-naturally occurring conjugate of interferon βand a non-polypeptide moiety, said treatment further giving rise tofewer adverse psychological effects than treatment with interferon β.The conjugate is preferably a conjugate of the invention.

[0351] Also contemplated is use of a nucleotide sequence encoding apolypeptide of the invention in gene therapy applications. Inparticular, it may be of interest to use a nucleotide sequence encodinga polypeptide as described in the section above entitled “GlycosylatedPolypeptides of the Invention modified to incorporate additionalglycosylation sites”. The glycosylation of the polypeptides is thusachieved during the course of the gene therapy, i.e. after expression ofthe nucleotide sequence in the human body.

[0352] Gene therapy applications contemplated include treatment of thosediseases in which the polypeptide is expected to provide an effectivetherapy due to its antiviral activity, e.g., viral diseases, includinghepatitis such as hepatitis C, and particularly HPV, or other infectiousdiseases that are responsive to interferon β or infectious agentssensitive to interferon β. Furthermore, the conjugate or polypeptide ofthe invention may be used in the treatment of chronic inflammatorydemyelinating polyradiculoneuropathy, and of severe necrotisingcutaneous lesions. Also, gene therapy in connection with the treatmentof any MS type is contemplated. Similarly, this invention contemplatesgene therapy applications for immunomodulation, as well as in thetreatment of those diseases in which interferon β is expected to providean effective therapy due to its antiproliferative activity, e.g., tumorsand cancers, or other conditions characterized by undesired cellproliferation, such as restenosis. A further description of such genetherapy is provided in WO 95/25170.

[0353] Local delivery of interferon β using gene therapy may provide thetherapeutic agent to the target area while avoiding potential toxicityproblems associated with non-specific administration.

[0354] Both in Vitro and in Vivo Gene Therapy Methodologies areContemplated.

[0355] Several methods for transferring potentially therapeutic genes todefined cell populations are known. For further reference see, e.g.,Mulligan, “The Basic Science Of Gene Therapy”, Science, 260, pp. 926-31(1993). These methods include:

[0356] Direct gene transfer, e.g., as disclosed by Wolff et al., “DirectGene transfer Into Mouse Muscle In vivo”, Science 247, pp. 1465-68(1990);

[0357] Liposome-mediated DNA transfer, e.g., as disclosed by Caplen etal., “Liposome-mediated CFTR Gene Transfer to the Nasal Epithelium OfPatients With Cystic Fibrosis” Nature Med., 3, pp. 39-46 (1995);Crystal, “The Gene As A Drug”, Nature Med., 1, pp.-15-17 (1995); Gao andHuang, “A Novel Cationic Liposome Reagent For Efficient Transfection ofMammalian Cells”, Biochem.Biophys Res. Comm., 179, pp. 280-85 (1991);

[0358] Retrovirus-mediated DNA transfer, e.g., as disclosed by Kay etal., “In vivo Gene Therapy of Hemophilia B: Sustained Partial CorrectionIn Factor IX-Deficient Dogs”, Science, 262, pp. 117-19 (1993); Anderson,“Human Gene Therapy”, Science, 256, pp.808-13(1992);

[0359] DNA Virus-mediated DNA transfer. Such DNA viruses includeadenoviruses (preferably Ad-2 or Ad-5 based vectors), herpes viruses(preferably herpes simplex virus based vectors), and parvoviruses(preferably “defective” or non-autonomous parvovirus based vectors, morepreferably adeno-associated virus based vectors, most preferably AAV-2based vectors). See, e.g., Ali et al., “The Use Of DNA Viruses asVectors for Gene Therapy”, Gene Therapy, 1, pp. 367-84 (1994); U.S. Pat.Nos. 4,797,368, and 5,139,941.

[0360] The invention is further described in the following examples. Theexamples should not, in any manner, be understood as limiting thegenerality of the present specification and claims.

MATERIALS AND METHODS

[0361] Materials

[0362] HeLa cells—(available from American Type Culture Collection(ATCC)

[0363] ISRE-Luc (Stratagene, La Jolla USA)

[0364] pCDNA 3.1/hygro (Invitrogen, Carlsbad USA)

[0365] pGL3 basic vector (Promega)

[0366] Human genomic DNA (CloneTech, USA)

[0367] DMEM medium: Dulbecco's Modified Eagle Media (DMEM), 10% fetalbovine serum (available from Life Technologies A/S, Copenhagen, Denmark)

[0368] Assays

[0369] Interferon Assay

[0370] It has previously been published that interferon β interacts withand activates Interferon type I receptors on HeLa cells. Consequently,transcription is activated at promoters containing an InterferonStimulated Response Element (ISRE). It is thus possible to screen foragonists of interferon receptors by use of an ISRE coupled luciferasereporter gene (ISRE-luc) placed in HeLa cells.

[0371] Primary Assay

[0372] HeLa cells are co-transfected with ISRE-Luc and pCDNA 3.1/hygroand foci (cell clones) are created by selection in DMEM media containingHygromycin B. Cell clones are screened for luciferase activity in thepresence or absence of interferon β. Those clones showing the highestratio of stimulated to unstimulated luciferase activity are used infurther assays.

[0373] To screen muteins, 15,000 cells/well are seeded in 96 wellculture plates and incubated overnight in DMEM media. The next daymuteins as well as a known standard are added to the cells in variousconcentrations. The plates are incubated for 6 hours at 37 C in a 5% CO₂air atmosphere LucLite substrate (Packard Bioscience, Groningen TheNetherlands) is subsequently added to each well. Plates are sealed andluminescence measured on a TopCount luminometer (Packard) in SPC (singlephoton counting) mode. Each individual plate contains wells incubatedwith interferon β as a stimulated control and other wells containingnormal media as an unstimulated control. The ratio between stimulatedand unstimulated luciferase activity serves as an internal standard forboth mutein activity and experiment-to-experiment variation.

[0374] Secondary Assay

[0375] Currently, there are 18 non-allelic interferon α genes and oneinterferon β gene. These proteins exhibit overlapping activities andthus it is critical to ensure that muteins retain the selectivity andspecificity of interferon β.

[0376] The β-R1 gene is activated by interferon β but not by otherinterferons. The transciption of β-R1 thus serves as a second marker ofinterferon β activation and is used to ensure that muteins retaininterferon β activity. A 300 bp promoter fragment of β-R1 shown to driveinterferon sensitive transcription (Rani. M. R. et al (1996) JBC 27122878-22884) was isolated by PCR from human genomic DNA and insertedinto the pGL3 basic vector (Promega). The resulting β-R1: luciferasegene is used in assays similar to the primary assay described above. Inastrocytoma cells, the resulting β-R1: luciferase gene has beendescribed to show 250 fold higher sensitivity to interferon β than tointerferon α (Rani et al. op cit).

[0377] Elisa Assay

[0378] The concentration of IFN-β is quantitated by use of a commercialsandwich immunoassay (PBL Biomedical Laboratories, New Brunswick, N.J.,USA). The kit is based on an ELISA with monoclonal mouse anti-IFN-βantibodies for catching and detection of IFN-β in test samples. Thedetecting antibody is conjugated to biotin.

[0379] Tests samples and recombinant human IFN-β standard are added in0.1 mL in concentrations from 10-0.25 ng/mL to microtiter plates,precoated with catching antibody. The plates are incubated at RT for 1hr. Samples and standard are diluted in kit dilution buffer.

[0380] The plates are washed in the kit buffer and incubated with thebiotinylated detecting antibody in 0.1 mL for 1 hr at RT. After anotherwash the streptavidin-horseradishperoxidase conjugate is added in 0.1 mLand incubated for 1 hr at RT.

[0381] The reaction is visualised by addition of 0.1 mLTetramethylbenzidine (TMB) substrate chromogen. The plates are incubatedfor 15 minutes in the dark at RT and the reaction is stopped by additionof stop solution. The absorbanse is read at 450 nm using an ELISAreader.

[0382] Receptor Binding Assay

[0383] The receptor binding capability of a polypeptide or conjugate ofthe invention can be determined using the assay described in WO 95/25170entitled “Analysis Of IFN-β(Phe₁₀₁) For Receptor Binding” (which isbased on Daudi or A549 cells). Soluble domains of IFNAR1 anid IFNAR2 canbe obtained essentially as described by Arduini et al, Protein Science,1999, vol. 8, 1867-1877 or as described in Example 9 herein.

[0384] Alternatively, the receptor binding capability is determinedusing a crosslinking agent such as disuccinimidyl suberate (DSS)available from Pierce, Rockford, Ill., USA as follows:

[0385] The polypeptide or conjugate is incubated with soluble IFNAR-2receptor in the presence or absence of DSS in accordance with themanufacturer's instructions. Samples are separated by SDS-PAGE, and awestern blot using anti-interferon β or anti-IFNAR2 antibodies isperformed. The presence of a functional interferon βpolypeptide/conjugate: receptor interaction is apparent by an increasein the molecular size of receptor and interferon β in the presence ofDSS.

[0386] Furthermore, a crosslinking assay using a polypeptide orconjugate of the invention and both receptor subunits (IFNAR-1 andIFNAR-2) can establish Interferon receptor 1 binding ability. In thisconnection it has been published that IFNAR-1 binds only after aninterferon β: IFNAR-2 complex is formed (Mogensen et al., Journal ofInterferon and Cytokine Research, 19:1069-1098, 1999).

[0387] In Vitro Immunogenicity Tests of Interferon β Conjugates

[0388] Reduced immunogenicity of a conjugate or polypeptide of theinvention is determined by use of an ELISA method measuring theimmunoreactivity of the conjugate or polypeptide relative to a referencemolecule or preparation. The reference molecule or preparation isnormally a recombinant human interferon β preparation such as Avonex,Rebif or Betaseron, or another recombinant human interferon βpreparation produced by a method equivalent to the way these productsare made. The ELISA method is based on antibodies from patients treatedwith one of these recombinant interferon β preparations. Theimmunogenicity is considered to be reduced when the conjugate orpolypeptide of the invention has a statistically significant lowerresponse in the assay than the reference molecule or preparation.

[0389] Another method of determining immunogenicity is by use of serafrom patients treated with interferon beta (i.e. any commercialinterferon β product) in an analogous manner to that described by Rosset al. J. Clin Invest. 95, 1974-78, 1995. In the antiviralneutralisation bioassay reduced immunogenicity results in reducedinhibition of a conjugate of the invention by patient sera compared to awt IFN-beta reference molecule. Furthermore, in the biochemical IFNbinding assay a less immunogenic conjugate is expected to bind topatient IgG to a lesser extent than reference IFN-beta molecules.

[0390] For the neutralisation assay, the reference and conjugatemolecules are added in a concentration that produces approximately 80%virus protection in the antiviral neutralisation bioassay. The IFN-βproteins are mixed with patient sera in various dilutions (starting at1:20).

[0391] Antiviral Activity

[0392] The antiviral bioassay is performed using A549 cells (CCL 185,American tissue culture collection) and Encephalomyocarditis (EMC) virus(VR-129B, American tissue culture collection).

[0393] The cells are seeded in 96 well tissue culture plates at aconcentration of 10,000 cells/well and incubated at 37° C. in a 5% CO₂air atmosphere. A polypeptide or conjugate of the invention is added inconcentrations from 100-0.0001 IU/mL in a total of 100 μl DMEM mediumcontaining fetal calf serum and antibiotics.

[0394] After 24 hours the medium is removed and 0.1 mL fresh mediumcontaining EMC virus is added to each well. The EMC virus is added in aconcentration that causes 100% cell death in IFN-β free cell culturesafter 24 hours.

[0395] After another 24 hrs, the antiviral effect of the polypeptide orconjugate is measured using the WST-1 assay. 0.01 mL WST-1 (WST-1 cellproliferation agent, Roche Diagnostics GmbH, Mannheim, Germany) is addedto 0.1 mL culture and incubated for ½-2 hours at 37° C. in a 5% CO₂ airatmosphere The cleavage of the tetrazolium salt WST-1 by mitochondrialdehydrogenases in viable cells results in the formation of formazan thatis quantified by measuring the absorbance at 450 nm.

[0396] Neutralisation of Activity in Interferon Stimulated ResponseElement (ISRE) Aassay

[0397] The interferon β neutralising effect of anti-interferon β seraare analysed using the ISRE-Luciferase activity assay.

[0398] Sera from interferon β treated patients or from immunised animalsare used. Sera are added either in a fixed concentration (dilution1:20-1:500 (pt sera) or 20-600 ng/mL (animal sera)) or in five-foldserial dilutions of sera starting at 1/20 (pt sera) or 600 ng/mL (animalsera). Interferon β is added either in five fold-dilutions starting at25.000 IU/mL or in a fixed concentration (0.1-10 IU/mL) in a totalvolume of 80 μl DMEM medium+10% FCS. The sera are incubated for 1 hr. at37° C. with IFN-β.

[0399] The samples are then transferred to 96 well tissue culture platescontaining HeLa cells transfected with ISRE-Luc grown from 24 hrs before(15,000 cells/well) in DMEM media. The cultures are incubated for 6hours at 37° C. in a 5% CO₂ air atmosphere. LucLite substrate (PackardBioscience, Groningen, The Netherlands) is subsequently added to eachwell. Plates are sealed and luminescence measured on a TopCountluminometer (Packard) in SPC (single photon counting) mode.

[0400] When interferon β samples are titrated in the presence of a fixedamount of serum, the neutralising effect was defined as fold inhibition(FI) quantified as EC50(w. serum)/EC50 (w/o serum). The reduction ofantibody neutralisation of interferon β variant proteins is defined as$\left( {1 - \frac{{FI}\quad {variant}}{{FI}\quad {wt}}} \right) \times 100\quad \%$

[0401] Biological Half-Life Measurement of a PEG-Interferon β Conjugate

[0402] Measurement of biological half-life can be carried out in anumber of ways described in the literature. One method is described byMunafo et al (European Journal of Neurology 1998, vol 5 No2 p 187-193),who used an ELISA method to detect serum levels of interferon β aftersubcutaneous and intramuscular administration of interferon β.

[0403] The rapid decrease of interferon β serum concentrations afteri.v. administration has made it important to evaluate biologicalresponses to interferon β treatment. However it is contemplated that theconjugates of the present invention will have prolonged serum half lifesalso after i.v. administration making it possible to measure by e.g. anELISA method or by the primary screening assay.

[0404] Different pharmacodynamic markers (e.g. serum neupterin and beta2microglobulin) have also been studied (Clin Drug Invest (1999)18(1):27-34). These can equally well be used to evaluate prolongedbiological effect. These experiments may also be carried out in suitableanimal species, e.g. rats.

[0405] Assays to assess the biological effects of interferon β such asantiviral, antiproliferative and immunomodulatory effects (as describedin e.g. Annals of Neurology 1995 vol 37 No 1 p 7-15) can be usedtogether with the primary and secondary screening assays describedherein to evaluate the biological efficacy of the conjugate incomparison to wild type interferon β.

[0406] Finally an animal model such as the commonly used experimentalautoimmune encephalomyelitis (EAE) model can be used to establishefficacy of a conjugate or polypeptide of the invention. In the EAEmodel immunization with myelin or myelin derived proteins elicits adisease mimicking the majority of the inflammatory and neurologicfeatures of multiple sclerosis in humans. EAE has been used in mice,rats, rabbits, and marmosets {Cannella, Hoban, et al. 1998 ID: 695}{Zaprianova, Deleva, et al. 1997 ID: 699} {Hassouna, Galeano, et al.1983 ID: 731} {Genain & Hauser 1997 ID: 703}. Other models includeTheiler's murine encephalomyelitis virus (TMEV) model {Murray, McGavern,et al. 1998 ID: 694}. will be used to establish efficacy of theinterferon β conjugate.

[0407] PEGylation in Microtiter Plates of a Tagged Polypeptide withInterferon β Activity

[0408] The Method Comprises

[0409] Expressing the interferon β polypeptide with a suitable tag, e.g.any of the tags exemplified in the general description above.

[0410] Transferring culture broth to one or more wells in a microtiterplate capable of immobilising the tagged polypeptide. When the tag isHis-His-His-His-His-His (Casey et al, J. Immunol. Meth., 179, 105(1995)), a Ni-NTA HisSorb microtiter plate commercially available fromQiaGen can be used.

[0411] After allowing for immobilising the tagged polypeptide to themicrotiter plate, the wells are washed in a buffer suitable for bindingand subsequent PEGylation.

[0412] Incubating the wells with the activated PEG of choice. As anexample, M-SPA-5000 from Shearwater Polymers is used. The molar ratio ofactivated PEG to polypeptide has to be optimised, but will typically begreater than 10:1 more typically greater than 100:1.

[0413] After a suitable reaction time at ambient temperature, typicallyaround 1 hour, the reaction is stopped by removal of the activated PEGsolution. The conjugated protein is eluted from the plate by incubationwith a suitable buffer. Suitable elution buffers may contain Imidazole,excess NTA or another chelating compound.

[0414] The conjugated protein is assayed for biological activity andimmunogenicity as appropriate.

[0415] This tag may optionally be cleaved off using a method known inthe art, e.g. using diaminopeptidase and the Gln in pos −1 will beconverted to pyroglutamyl with GCT (glutamylcyclotransferase) andfinally cleaved off with PGAP (pyro-glutamyl-aminopeptidase) giving thenative protein. The process involves several steps of metal chelateaffinity chromatography. Alternatively, the tagged polypeptide may beconjugated.

[0416] PEGylation of a Receptor-Bound Interferon β Polypeptide

[0417] In order to optimize PEGylation of an interferon β polypeptide ina manner excluding PEGylation of lysines involved in receptorrecognition, the following method has been developed: The solubledomains of IFNAR1 and IFNAR2 are obtained essentially as described inArduini et al, Protein Science (1999), vol 8: 1867-1877 or as describedin Example 9.

[0418] A ternary complex consisting of an interferon β polypeptide, asoluble domain of IFNAR1 and a soluble domain of IFNAR2 in a 1:1:1stoichiometry is formed in a PBS buffer at pH 7-9. The concentration ofInterferon β polypeptide is approximately 20 ug/ml or 1 uM and thereceptors are present at equimolar concentration.

[0419] M-SPA-5000 from Shearwater Polymers, Inc is added at 3 differentconcentration levels corresponding to 5, 20 or 100 molar excess ofinterferon β polypeptide. The reaction time is 30 min at RT. After the30 min reaction period, the pH of the reaction mixture is adjusted to pH2.0 and the reaction mixture is applied to a Vydac C18 column and elutedwith an acetonitrile gradient essentially as described (Utsumi etal, J.Biochem., vol 101, 1199-1208, (1987). Alternatively and more elegantly,an isopropanol gradient can be used.

[0420] Fractions are analyzed using the primary screening assaydescribed herein and active PEGylated interferon-β polypeptide obtainedby this method stored at −80 C in PBS, pH 7 containing 1 mg/ml HSA.

[0421] Alternatively, to the procedure described above a soluble domainof IFNAR2 is used as the only receptor component to form a binarycomplex. Furthermore, IFNAR2 may be immobilized on a suitable resin(e.g. Epoxy activated Sepharose 6B) according to the manufacturesinstructions prior to forming the binary complex. After PEGylation, thePEGylated Interferon-β is eluted with a 0.1 M Glycin, pH 2 buffer andactivity measured as described after pH adjustment to neutral.

[0422] Accessible Surface Area (ASA)

[0423] The computer program Access (B. Lee and F. M.Richards, J.Mol.Biol. 55: 379-400 (1971)) version 2 (Copyright (c) 1983 YaleUniversity) are used to compute the accessible surface area (ASA) of theindividual atoms in the structure. This method typically uses aprobe-size of 1.4 Å and defines the Accessible Surface Area (ASA) as thearea formed by the centre of the probe. Prior to this calculation allwater molecules and all hydrogen atoms are removed from the coordinateset, as are other atoms not directly related to the protein. Alternativeprograms are available for computing ASA, e.g. the program WhatlfG.Vriend, J. Mol. Graph. (1990) 8, 52-56, electronically available atthe WWW interface onhttp://swift.embl-heidelberg.de/servers2/(R.Rodriguez et.al. CABIOS(1998) 14, 523-528.) using the option Accessibility to calculate theaccessible molecular surface.

[0424] Fractional ASA of Side Chain

[0425] The fractional ASA of the side chain atoms is computed bydivision of the sum of the ASA of the atoms in the side chain with avalue representing the ASA of the side chain atoms of that residue typein an extended ALA-×-ALA tripeptide. See Hubbard, Campbell & Thornton(1991) J.Mol.Biol.220,507-530. For this example the CA atom is regardedas a part of the side chain of Glycine residues but not for theremaining residues. The following table indicates the 100% ASA standardfor the side chain: Ala  69.23 Å² Arg 200.35 Å² Asn 106.25 Å² Asp 102.06Å² Cys  96.69 Å² Gln 140.58 Å² Glu 134.61 Å² Gly  32.28 Å² His 147.00 Å²Ile 137.91 Å² Leu 140.76 Å² Lys 162.50 Å² Met 156.08 Å² Phe 163.90 Å²Pro 119.65 Å² Ser  78.16 Å² Thr 101.67 Å² Trp 210.89 Å² Tyr 176.61 Å²Val 114.14 Å²

[0426] Determining Surface Exposed Amino Acid Residues

[0427] The three-dimensional crystal structure of human interferon betaat 2.2 Å resolution (Karpusas et al. Proc. Nat. Acad. Sci. USA (1997)94:11813-11818 is available from the Protein Data Bank (PDB) (Bernsteinet.al. J. Mol. Biol. (1977) 112 pp. 535) and electronically availablevia The Research Collaboratory for Structural Bioinformatics PDB athttp://www.pdb.org/ under accession code 1AU1. This crystal structurecontain two independent molecules of human interferon beta in thisexample the A molecule is used.

[0428] Surface Exposure:

[0429] Using the Whatlf program as described above the followingresidues were found to have zero surface accessibility for their sidechain atoms (for Gly the accessibility of the CA atom is used): G7, N14,C17, L21, I44, A55, A56, T58, 159, M62, L63, L98, L122,Y125, I129, L133,A142, W143, V146, I150, N153, I157, L160, T161, and L164.

[0430] Fractional Surface Exposure

[0431] For further analysis it was necessary to remodel the side chainsof residues R7 1, R113, K115, L116, M117 due to steric clashes. Theremodelling was done using Modeler 98, MSI INC. Performing fractionalASA calculations using the Access computer program on the remodelledinterferon beta molecule (only including the amino acid residues andexcluding the N-linked sugar moiety) resulted in the following residueshaving more than 25% of their side chain exposed to the surface: S2, N4,L5, F8, L9, R11, S12, F15, Q16 Q18, K19, W22, Q23, G26, R27, L28, E29,Y30, L32, K33, R35, M36, N37, D39, E42, K45, Q46, L47, Q48, Q49, Q51,K52, Q64, A68, R71, Q72, D73, S75, S76, G78, N80, E81, T82, E85, N86,A89, Y92, H93, N96, H97, K99, T100, E103, E104, K105, E107, K108, E109,D110, F111, R113, G114, K115, L116, S119, L120, H121, K123, R124, G127,R128, L130, H131, K134, A135, K136, E137, Y138, S139, H140, V148, R152,Y155, N158, G162, Y163, R165, and N166 and the following residues havemore than 50% of their side chain exposed to the surface: N4, L5, F8,S12, F15, Q16, K19 W22, G26, R27, E29, Y30, K33, R35, N37, D39, E42,Q46, Q48, Q49, Q51, K52, R71, D73, S75, G78, N80, E81, T82, E85, N86,A89, Y92, H93, K99, T100, E103, E104, E107, K108, D110, F111, L116,K123, R124, G127, H131, K134, E137, V148, Y155, R165, and N166.

ExampleS Example 1

[0432] Design of an Expression Cassette for Expression of Interferon βin Mammalian and Insect Cells

[0433] The DNA sequence, GenBank accession number M28622 (shown in SEQID NO 1), encompassing a full length cDNA encoding human interferon βwith its native signal peptide, was modified in order to facilitate highexpression in mammmalian cells. First the ATG start codon context wasmodified according to the Kozak consensus sequence (Kozak, M. J Mol BiolAug. 20, 1987; 196(4):947-50), such that there is a perfect match to theconsensus sequence upstream of the ATG start codon. Secondly the codonsof the native human interferon β was modified by making a bias in thecodon usage towards the codons frequently used in highly expressed humangenes. Subsequently, certain nucleotides in the sequence weresubstituted with others in order to introduce recognition sites for DNArestriction endonucleases (this allows for easier modification of theDNA sequence later). Primers were designed such that the gene could besynthesised: CBProFpr1 SEQ ID 35′GGCTAGCGTTTAAACTTAAGCTTCGCCACCATGACCAACAAGTGCCTGCTCCAGATCGCCCTGCTCCTGT-3′, CBProFpr2 SEQ ID 45′ACAACCTGCTCGGCTTCCTGCAGAGGAGTTCGAACTTCCAGTGCCAGAAGCTCCTGTGGCAGCTGAACGG-3′, CBProFpr3 SEQ ID 55′GAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCAGTTCCAGAAGGAGGACGCCGCTCTGACCATC-3′, CBProFpr4 SEQ ID 65′TTCCGCCAGGACTCCAGCTCCACCGGTTGGAACGAGACCATCGTGGAGAACCTGCTGGCCAACGTGTACC-3′, CBProFpr5 SEQ ID 75′AGGAGAAGCTGGAGAAGGAGGACTTCACCCGCGGCAAGCTGATGAGCTCCCTGCACCTGAAGCGCTACTA-3′, CBProFpr6 SEQ ID 85′GGAGTACAGCCACTGCGCCTGGACCATCGTACGCGTGGAGATCCTGCGCAACTTCTACTTCATCAACCGC-3′, CBProFpr9 SEQ ID 95′CACCACACTGGACTAGTGGATCCTTATCAGTTGCGCAGGTAGCCGGTCAGGCGGTTGATGAAGTAGAAGT-3′, CBProFpr10 SEQ ID 105′AGGCGCAGTGGCTGTACTCCTTGGCCTTCAGGTAGTGCAGGATGCGGCCATAGTAGCGCTTCAGGTGCAG-3′, CBProFpr11 SEQ ID 115′CTCCTTCTCCAGCTTCTCCTCCAGCACGGTCTTCAGGTGGTTGATCTGGTGGTACACGTTGGCCAGCAGG-3′, CBProFpr12 SEQ ID 125′GAGCTGGAGTCCTGGCGGAAGATGGCGAAGATGTTCTGCAGCATCTCGTAGATGGTCAGAGCGGCGTCCT-3′, CBProFpr13 SEQ ID 135′CCTCGGGGATGTCGAAGTTCATCCTGTCCTTCAGGCAGTACTCCAGGCGCCCGTTCAGCTGCCACAGGAG-3′, CBProFpr14 SEQ ID 145′CAGGAAGCCGAGCAGGTTGTAGCTCATCGATAGGGCCGTGGTGCTGAAGCACAGGAGCAGGGCGATCTGG-3′.

[0434] The primers were assembled to the synthetic gene by one step PCRusing Platinum Pfx-polymerase kit (Life Technologies) and standard threestep PCR cycling parameters. The assembled gene was amplified by PCRusing the same conditions.

[0435] A cDNA encoding a N-terminal extended form of human interferon βwas synthesised using the same PCR conditions as described above butwith the primers CBProFpr1 and −14 substituted with the primers:CBProFpr7 SEQ ID 15 5′CTGCTCCAGATCGCCCTGCTCCTGTGCTTCAGCACCACGGCCCTATCGATGAAGCACCAGCACCAGCATC-3′, CBProFpr8 SEQ ID 165′CACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAAC-3′, CBProFpr15 SEQ ID 175′CAGGAAGCCGAGCAGGTTGTAGCTCATCTGTTGGTGTTGATGTTGGTGCTGATGCTGGTGCTGGTGCTTC-3′, CBProFpr16 SEQ ID 185′AGCAGGGCGATCTGGAGCAGGCACTTGTTGGTCATGGTGGCGAAGCTTAAGTTTAAACGCTAGCCAGCTT-3′,

[0436] in order to incorporate a purification TAG in the interferon βmolecule.

[0437] The synthesised genes were cloned into pcDNA3.1/Hygro(Invitrogen) between the HindIII site at the 5′ end and the BamHI at the3′, resulting in pCBProF1 and pCBProF2.

[0438] The synthetic intron from pCI-Neo (Promega) was amplified usingstandard PCR conditions as described above and the primers: CBProFpr37SEQ ID 19 5′-CCGTCAGATCCTAGGCTAGCTTATTGCGGTAGTTTATCAC-3′, CBProFpr38 SEQID 20 5′-GAGCTCGGTACCAAGCTTTTAAGAGCTGTAAT-3′,

[0439] resulting in a 332 bp PCR fragment which was cut with NheI andHindIII and inserted in the 5′UTR of the plasmids pCBProF1 and pCBProF2resulting in pCBProF4 and pCBProF5.

[0440] Codons for individual amino acids were changed by amplifyingrelevant regions of the coding region by PCR in such a way that the PCRintroduced changes in the sequence can be introduced in the expressionplasmids by classical cloning techniques. E.g. the primers:Lys45arg-5′primer (NarI/KasI): SEQ ID 215′GCTGAACGGGCGCCTGGAGTACTGCCTGAAGGACAGGATGAACTTCGACATCCCCGAGGAAATCCGCCAGCTGCAGC-3′, Lys45mut-3′primer (BsiWI): SEQ ID 225′TCTCCACGCGTACGATGGTCCAGGCGCAGTGGCTG-3′,

[0441] were used to introduce a K45R substitution in the PCR-fragmentspanning the region from position 1055 to 1243 in pCBProF1. Both the PCRfragment and pCBProF1 was cut with NarI and BsiWI which are both unique.The PCR fragment and the vector backbone of pCBProF1 are purified andligated resulting in substitution of the Lys45 codon AAG with the Argcodon CGC in pCBProF1.

[0442] Furthermore, SOE (sequence overhang extension) PCR was used forintroduction of amino acid substitutions. In the SOE-PCR both theN-terminal part and the C-terminal part of the INFB molecule were firstamplified in individual primary PCRs.

[0443] For these primary PCRs the central complementary primers weresynthesised such that the codon(s) for the amino acid(s) to besubstituted is/are changed to the desired codon(s). The terminal primerswere standard primers defining the N- and C-terminal of the INFβmolecule respectively. Further the terminal primers provided arestriction enzyme site enabling subsequent cloning of the full-lengthPCR product. Thus, the central (nonsense) primer and the N-terminal(sense) primer were used to amplify the N-terminal part of the INFβcoding region in one of the primary PCRs and equivalently for theC-terminal part. Once amplified the N- and C-terminal parts areassembled into the full-length product in a secondary PCR and clonedinto a modified version of pCDNA3.1/Hygro as described above. Forinstance, the following primers were used to introduce the mutations forthe substitutions F111N and R113T: CBProFprimer9(Sense): (SEQ ID NO 23)CACCACACTGGACTAGTGGATCCTTATCAGTTGCGCAGGTAGCCGGTCAG GCGGTTG ATGAAGTAGAAGT, CBProFprimer231(Antisense): (SEQ ID NO 24)CATCAGCTTGCCGGTGGTGTTGTCCTCCTTC, CBProFprimer230 (Sense): (SEQ ID NO 25)GAAGGAGGACAACACCACCGGCAAGCTGATG, CBProFprimer42 (Antisense): (SEQ ID NO26) CACACTGGACTAGTAAGCTTTTATCAGTTGCGCAGGTAGC,

[0444] Furthermore, in cases where the introduced mutation(s) weresufficiently close to a unique restriction endo-nuclease site in theexpression plasmid variant genes were constructed using constructionprocedure encompassing a single PCR step and a subsequent cloning. Forinstance, the substitution K19R was introduced by use of the PCR primer:

[0445] CBProFpr58:

[0446] GAGGAGTTCGAACTTCCAGTGCCAGCGCCTCCTGTGGCAGCTGAACG (SEQ ID NO 27),and CBProFprimer9:

[0447] The PCR product was subsequently cloned using the restrictionendo-nuclease sites BsiWI and BstBI.

Example 2

[0448] Expression of Human Interferon β in a Baclovirus/Insect CellSystem

[0449] In order to express the synthetic gene, encoding human interferonβ harboured in pCBProF 1 (described in example 1) in thebaculovirus/insect cell system the gene was excised with NheI and XhoIand ligated into the transfer vector pBlueBac 4.5, which is included inthe MaxBac 2.0 Transfection kit obtained from Invitrogen (San Diego,USA). All methods used for generation of recombinant baculovirus andexpression in insect cells are described in the “MaxBac 2.0 Transfectionand Expression Manual” included in the kit.

[0450] In brief, together with liniarized AcMNPV DNA (Bac-N-Blue DNA)pBlueBac 4.5-interferon β CBProF1) was transfected into SF9 cells. 3days post-transfection the transfection supernatant was harvested and aplaque assay with appropriate viral dilutions was prepared. Bluedistinct plaques were visible after 7 days and 6 individual plaques werecollected for propagation in a 6-well plate. After 5 days 2 ml virussupematant (P-1 stock) was harvested from each well. 0.75 ml was takenout from the P1 stocks and viral genomic DNA was isolated. The viralgenomic DNA's were analysed in PCR reactions with forward/reverseprimers in order to be able to select the recombinant baculovirusesamong the six P-1 stocks. A small aliquot from the recombinant P-1 stockwas tested in a human interferon β specific ELISA (available from PBLBiomedical Laboratories) in order to ensure that recombinant humaninterferon β was present in the supernatant.

[0451] For further propagation of chosen recombinant baculovirus 6×10⁶SF9 cells were seeded in a T-80 culture flask and infected with 200 μlof the P-1 stock. After 5 days the supernatant (P-2 stock) was harvestedand 2 ml of the P-2 stock was used to infect a 100 ml suspension culture(1×10⁶ SF9 cells/ml) in a 500 ml Erlenmeyer flask (Coming). After 5 daysthe supernatant (P-3 stock) was harvested and the virus titer wasdetermined by plaque assay.

[0452] In order to produce human interferon β for purification 1×10⁹ SF9cells were harvested from a backup suspension culture. In a 50 mlscrew-cap tube the SF9 cells were infected with recombinant baculovirusfrom the P-3 stock (MOI=2) in a period of 15 minutes. Hereafter thecells were spun down and washed one time in serum-free medium (Sf-900 IISFM, Gibco BRL) and transferred to a 2800 ml Triple Baffle Fembach Flask(Bellco) containing 11 serum-free medium. 3 days post-infection themedium supernatant was harvested and the recombinant human interferon βwas purified.

[0453] Purification of Interferon β Molecules

[0454] The fermentation broth is concentrated and/or pH adjusted toapproximately 4.5 after dilution to suitable ionic strength. Suitable isintended to mean that the ionic strength is so low that interferon βwill bind to a Mono S cation exchange column (Pharmacia) equilibrated in4 mM acetic acid pH 4.5 (buffer A). After application, the column iswashed with 3 column volumes buffer A and interferon β is cluted with alinear gradient from buffer A to buffer A including 1 M NaCl.Alternatively purification can be obtained as described for Interferon α(Analytical Biochemistry 247, 434-440 (1997) using a TSK-gel SP-5PWcolumn (Toso Haas)).

[0455] Alternatively His tagged interferon β can be purified using IMAC(Immobilized Metal Affinity Chromatography) in accordance with wellknown methods, e.g., as described by UniZyme Laboratories, Denmark.

[0456] Another purification method makes use of monoclonal or polyclonalantibodies. Interferon β fermentation broth is adjusted to pH 7 and 0.5M NaCl and applied to a column with immobilized monoclonal antibody torecombinant human interferon β. The column is equilibrated with e.g. 10mM Tris, 0.5 M NaCl, pH 7 (Buffer B) prior to application. Afterapplication the column is washed with 3 column volumes Buffer B andeluted with a suitable buffer at low pH (e.g. pH 2-3).

[0457] Alternatively, if the interferon β is tagged with e.g. the c-Mycpeptide (EQKLI SEEDL), monoclonal antibodies raised against the c-Mycpeptide, can be used in a similar fashion. Immobilization of antibody tothe column is achieved using e.g. CNBr-Sepharose (Pharmacia) accordingto the manufacturers instructions.

[0458] A combination of Cation exchange chromatography, IMAC and/orantibody chromatography may be applied if necessary to obtain relevantpurity for further experiments.

[0459] Purity, identity, quantity and activity of eluted fractions fromthe abovementioned columns can be determined using a combination ofmethods known by the person skilled in the art. These may include one ormore of the following assays and methods or other relevant methods knownby the person skilled in the art: the primary and secondary assaysdescribed above, ELISA methods, SDS-PAGE, western blotting, IEF, HPLC,amino acid sequencing, mass spectrometry and amino acid analysis.

[0460] Following purification, the modified interferon β polypeptide maybe subjected to conjugation to a polymer molecule such as M-SPA-5000from Shearwater Polymers according to the manufacturer's instructions.Preferably, the receptor recognition site of the purified modifiedinterferon β polypeptide is blocked prior to conjugation as described inthe Materials and Methods section herein.

Example 3

[0461] Expression of Human Interferon β in HEK293 Cells

[0462] In order to express the synthetic gene, encoding human interferonβ, harboured by pCBProF1 (described in example 1), in HEK293 cells (ATCCCat. No. CRL-1573) the gene was PCR-amplified with the two primers PBR 7(5′-CGCGGATCCATATGACCAACAAGTGCCTG-3′) (SEQ ID NO 28) and PBR 2(5′-CGCGGATCCTTATCAGTTGCGCAG-3′) (SEQ ID NO 29) and cloned into theBamHI site of pcDNA3.1 (−) (Invitrogen, USA) in correct orientation,giving the plasmid pPR9.

[0463] For transfection of the HEK293 cell line a T-25 culture flask wasseeded to 50% confluency in DMEM medium (Life Technologies, USA)containing 10% FBS and incubated over night. By usage of FuGENE 6Transfection Reagent (Roche, USA) pPR9 was transfected into the cells:To 95 μl serum-free DMEM medium was added 5 μl FuGENE 6 and 1.7 μl (2μg) pPR9 and incubated at room temperature for 20 minutes. Thetransfection complex was then added drop-wise to the cells and theculture flask was returned to the incubator. Next day the cells weretrypsinized and seeded into a T-80 culture flask in DMEM mediumcontaining 10% FBS and 500 μg Geneticin (Life Technologies) per ml.

[0464] At confluency it was confirmed, by usage of a human interferon βspecific ELISA, that the primary transfection-pool was expressing thewished protein and the cells were sub-cloned by limited dilution. Inthis way a high-producing HEK293 clone was identified expressing humaninterferon β.

Example 4

[0465] High Level Expression of Interferon β CHO Cells

[0466] The cell line CHO K1 [p22]-E4 (ATCC # CCL-61) stably expressinghuman interferon β was passed 1:10 from a confluent culture andpropagated as adherent cells in T-25 flasks in serum containing medium(MEMα w/ribonucleotides and deoxyribonucleotides (Gibco/BRL Cat #32571), 10% FCS (Gibco/BRL Cat # 10091), penicillin and streptomycin(Gibco/BRL Cat # 15140-114) until confluence. The media was then changedto serum free media (RenCyte CHO; MediCult Cat.# 22600140) for 24 hoursbefore including 5 mM Sodium Butyrate (Merck Cat # 8.17500) during amedium change. The cells were then allowed to express interferon β for48 hours prior to harvest of the medium. The interferon β concentrationin the duplicate cultures were deternined to be 854,797 IU/ml (withlower and upper 95% confidence interval at 711,134 IU/ml and 1,032,012IU/ml) respectively).

[0467] In a separate set of experiments, the cell line CHO K1 [p22]-E4stably expressing human interferon β was passed 1:10 from a confluentculture and propagated as adherent cells in serum containing medium(MEMα w/ribonucleotides and deoxyribonucleotides (Gibco/BRL Cat #32571), 10% FCS (Gibco/BRL Cat # 10091), penicillin and streptomycin(Gibco/BRL Cat # 15140-114) until confluence in a 10 layer cell factory(NUNC #165250). The media was then changed to serum free media; DMEM/F12(Gibco/BRL # 11039-021) with the addition of 1:100 ITS-A (Gibco/BRL #51300-044) and 1:500 EX-CYTE VLE (Serological Proteins Inc. # 81-129-1)and 1:100 penicillin and streptomycin (Gibco/BRL Cat # 15140-114) for 48hours before changing the medium with the further addition of 5 mMbutyrate (Merck Cat # 8.17500). The cells were then allowed to expressinterferon β for 48 hours prior to harvest of the medium. The interferonβ concentration was determined to be 824,791 IU/ml (with lower and upper95% confidence interval at 610,956 IU/ml and 1,099,722 IU/ml)respectively).

[0468] It is contemplated that interferon β polypeptides of theinvention may be produced in equally high yields in the same manner asany of those described above.

Example 5

[0469] Construction and Expression of Interderon β Variant with OneIntroduced Glycosylation Site

[0470] In order to insert an extra N-linked glycosylation site atposition 111 in HINF-β, the synthetic gene (hinf-β) encoding hINF-β(described in example 1) was altered by site-directed PCR mutagenesis.Using BIO-X-ACT (Bioline, UK) and the plasmid PF050[hinf-β)/pcDNA3.1(−)Hygro/Intron (a derivative of pcDNA3.1 (−)Hygro(Invitrogen, USA) in which a chimeric intron obtained from pCI-neo(Promega, USA) had been inserted between the BamHI and NheI sites in theMCS of the vector] as template, two PCR reactions were performed withtwo overlapping primer-sets [CB41(5′-TTTAAACTGGATCCAGCCACCATGACCAACAAG-3′) (SEQ ID NO 30)/CB55(5′-CGGCCATAGT AGCGCTTCAGGTGCAGGGAGCTCATCAGCTTGCCGGTGGTGTTGTCCTCCTTC-3′)(SEQ ID NO 31) and CB42 (SEQ ID 26)/CB86(5′-GAAGGAGGACAACACCACCGGCAAGCTGATGAGCTCCCTGCACCTGAAGCGCTACT ATGGCCG-3′) (SEQ ID NO 32) resulting in two fragments of 446 and 184 basepairs, respectively. These two fragments were assembled in a third PCRwith the flanking primers CB41 and CB42. The resulting gene was insertedinto the mammalian expression vector pcDNA3.1 (−)Hygro/Intron andconfirmed by DNA sequencing to have the correct base changes leading tothe substitutions F111N and R113T in hINFβ (plasmid designated PF085).

[0471] To test the activity of the [F111N+R113T]hINF-β variant, PF085was transfected into the CHO K1 cell line (ATCC #CCL-61) by use ofLipofectamine 2000 (Life Technologies, USA) as transfection agent. 24hours later the culture medium was harvested and assayed for INF-βactivity/concentration: Activity: 56046 IU/ml [primary assay] ELISA:  80 ng/ml Specific activity:   7 × 10⁸ IU/mg

[0472] As seen, the [F111N+R113T]hINF-β variant has a very high specificactivity, about twice the specific activity of wt hINF-β.

Example 6

[0473] Construction and Expression of Interderon β with AnotherIntroduced Glycosylation Site [Q49N+Q51T]

[0474] Analogously to what is described in Example 5 an extra N-linkedglycosylation site was introduced in position 49 by means of thesubstitutions Q49N and Q51T. Using PF043 (hinf-β/pcDNA3.1 (Invitrogen,USA)) as template, two PCR reactions were performed with two overlappingprimer-sets [PBR7 (SEQ ID NO 28)/PBR78(5′-GGCGTCCTCCTTGGTGAAGTTCTGCAGCTG-3′) (SEQ ID NO 33) andPBR8.(5′-ATATATCCCAAGCTTTTATCAGTTGCGCAGGTAGCCGGT-3′) (SEQ ID NO34)/PBR77 (5′-CAGCTGCAGAACTTCACCAAGGAGGACGCC-3′) (SEQ ID NO 35)resulting in two fragments of 228 and 369 base pairs, respectively.These two fragments were assembled in a third PCR with the flankingprimers PBR7 and PBR8. The resulting gene was inserted into themammalian expression vector pcDNA3.1(−)Hygro/Intron and confirmed by DNAsequencing to have the correct base changes leading to [Q49N,Q51T]hINF-β(plasmid designated PF104).

[0475] To test the activity of the [Q49N+Q51T]hINF-β variant, PF104 wastransfected into the CHO K1 cell line by use of Lipofectamine 2000 (LifeTechnologies, USA) as transfection agent. 24 hours later the culturemedium was harvested and assayed for INF-β activity/concentration:Activity: 17639 IU/ml [primary assay] ELISA:   10 ng/ml Specificactivity:   1.7 × 10⁹ IU/mg

[0476] As observed here the [Q49N+Q51T]hINF-β variant has a highspecific activity.

[0477] This may be due to poor recognition by one of the monoclonalantibodies used in the ELISA.

Example 7

[0478] Construction and Expression of Interderon β with Two IntroducedGlycosylation Site

[0479] The additional glycosylation sites described in Examples 5 and 6were introduced into human interferon β by means of the substitutionsQ49N, Q51T, F111N, and R113T.

[0480] Using PF085 (described in example 5) as template, two PCRreactions were performed with two overlapping primer-sets [PBR89(5′CGCGGATCCAGCCACCATGACCAACAAGTGCCTG) (SEQ ID NO 36)/PBR78 (SEQ ID NO33) and PBR8 (SEQ ID NO 34) )/PBR77 (SEQ ID NO 35)] resulting in twofragments of 228 and 369 base pairs, respectively.

[0481] These two fragments were assembled in a third PCR with theflanking primers PBR89 and PBR8. The resulting gene was inserted intothe mammalian expression vector pcDNA3.1(−)Hygro/Intron and confirmed bysequencing to have the correct base changes leading to [Q49N, Q51T,F111N, R113T] hINF-β (plasmid designated PF123).

[0482] PF123 was transfected into CHO K1 cells by use of Fugene 6(Roche) as transfection agent. 24 hours later the culture medium washarvested and assayed for INF-β activity/concentration: Activity: 29401IU/ml [primary assay] ELISA:   14 ng/ml Specific activity:   2.1 × 10⁹IU/ml

[0483] As observed here the [Q49N+Q51T+F111N+R113T]hINF-β variant alsohas a high specific activity.

[0484] The variant was found to have receptor binding activity in thereceptor binding assay described in the Materials and Methods section,which is based on the use of the crosslinking agent DSS.

Example 8

[0485] Production of [Q49N+Q51T+F111N+R113T]Interferon-β GlycosylationVariant in Roller Bottles

[0486] A CHOK1 sub-clone (5/G-10) producing the [Q49N+Q51T+F111N+R113T]glycosylation variant was seeded into 2 roller bottles, each with anexpanded surface of 1700 cm² (Corning, USA), in 200 ml DMEM/F-12 medium(LifeTechnologies; Cat. # 31330) supplemented with 10% FBS andpenicillin/streptomycin (P/S). After 2 days the medium was exchanged.After another 2 days the two roller bottles were nearly 100% confluentand the medium was shifted to 300 ml serum-free UltraCHO medium (BioWhittaker; Cat. # 12-724) supplemented with 1/500 EX-CYTE (SerologicalsProteins; Cat. # 81129N) and P/S. Growing the cells in this mediumpromotes a higher cell mass, higher than can be achieved in the serumcontaining medium. After 2 days the medium was renewed. After another 2days the medium was shifted to the production medium: DMEM/F-12 medium(Life Technologies; Cat. # 21041) supplemented with 1/100 ITSA (LifeTechnologies; Cat. # 51300-044) [ITSA stands for Insulin (1.0g/L)—Transferrin (0.55 g/L)—Selenium (0.67 mg/L) supplement for Adherentcultures], 1/500 EC-CYTE and P/S. In the figure below is shown theproduction run where 300 ml medium was harvested from each roller bottleevery day. The harvested media from the two roller bottles were pooledbefore a medium sample was taken out for interferon β activitydetermination. As seen in FIG. 2 the production run was terminated after26 days. After a lag-period of 5 days the activity mediated by the[49N+Q51T+F111N+R113T]Interferon-β variant increased dramatically andfor the rest of the production run the harvested interferon-β activityper day, in average, was 2.4 million IU/ml×600 ml=1.440 billion IU. Intotal 3.2×10¹⁰ IU was produced corresponding to 160 mg protein (with ahypothetical specific activity of 2×10⁸ IU/mg).

Example 9

[0487] Production, Purification, and Pegylation of the Interferon-βVariant K19R+K45R+K123R

[0488] To end up with 100 ml serum-free medium containing theInterferon-β variant K19R+K45R+K123R, 3 T-175 flasks were seeded withCOS-7 cells in DMEM medium (Life technologies; Cat. # 21969-035)supplemented with 10% FBS plus Glutamine and penicillin/streptomycin. Onthe day of transfection (at nearly 100% confluency) the medium wasrenewed with 30 ml fresh medium 4-5 hours before the transfection. Toprepare the transfection, 1890 μl DMEM medium without supplements wasaliquoted into a 14 ml polypropylene tube (Coming). 210 μl Fugene 6(Roche) was added directly into the medium and incubated for 5 min atRT. In the meantime 168 μg plasmid DNA ([K19R, K45R,K123R]INF-β/pcDNA3.1(−)Hygro; PF #161) was aliquoted into another 14 mlpolypropylene tube. After 5 min incubation the Fugene 6 mix was addeddirectly to the DNA solution and incubated for 15 min at RT. Afterincubation about 700 μl was added drop wise to each of the three cellmedia.

[0489] Next day the transfection medium was substituted with 35 mlserum-free production medium. The serum-free medium is based on DMEMmedium (Life Technologies; Cat. # 31053-028) supplemented withGlutamine, Sodium Pyruvate, penicillin/streptomycin, 1% ITSA (LifeTechnologies; Cat. # 51300-044), and 0.2% Ex-Cyte (SerologicalsProteins; Cat. # 81-129). Before the production medium was added thecell layers were washed two times in the DMEM medium without additives.

[0490] Three days post-transfection the 100 ml serum-free medium washarvested for purification and PEGylation of the Interferon-β variant.pH was adjusted to 6.8 and conductivity adjusted to <10 mS/cm with MilliQ water. Then the broth was batch adsorbed to 1 ml SP 550 cationexchange resin (TosoHaas) preequilibrated with buffer A (20 mMphosphate, 100 mM NaCl, pH 7). After 2 h rotation end over end, theresin was allowed to sediment and transferred to a column. The resin waswashed with 5 column volumes buffer A and eluted with 2 ml buffer B (20mM phosphate, 800 mM NaCl, pH 7). The eluate was concentrated to 500 ulon VivaSpin (cutoff 10 kDa) after addition of 5% ethyleneglycol. Theconcentrate was adjusted to 50 mM phosphate, 0.3 M NaCl, 20%ethyleneglycol, pH 8 in a final volume of 2 ml and further concentratedto 0.5 ml.

[0491] The final concentrate was PEGylated as follows: to 100 ul of thefinal concentrate, 25 ul of activated mPEG-SPA (5000 kDa, Shearwater,Ala.) freshly prepared in phosphate buffer, pH 8 were added to makefinal concentrations of activated PEG of 0, 5, 10 , 25 or 50 mg/ml. Thereaction was allowed to proceed for 30 min at room temperature and thenquenched by addition of 50 mM glycine buffer. Samples were frozenimmediately at −80 C and bioactivity was measured as described (PrimaryAssay). Western blots of each sample were performed in order to evaluatethe amount of unreacted Interferon-β variant present in the PEGylatedsample.

[0492] Results demonstrate that at 25 mg activated PEG/ml, nonPEGylatedInterferon-β variant was absent as judged by western blot and thevariant retained 50% of its bioactivity compared to the control sample(treated identically, but with 0 mg/ml activated PEG).

Example 10

[0493] Expression and Purification of Soluble IFNAR2

[0494] The cDNA's encoding the extracellular domain of IFNAR-1 andIFNAR-2 (termed IFNAR1ec and IFNAR2ec, respectively) were amplified fromHeLa cell cDNA using PCR with primers corresponding to the first 10amino acid residues and the final 10 amino acid residues of theextracellular domain of IFNAR-2 (the nucleotide sequence of which isapparent from Novick et al., Cell, Vol. 77, pp 391-400, 1994) and thefirst 10 amino acid residues and the final 10 amino acid residues of theextracellular domain of IFNAR-1 (the nucleotide sequence of which isapparent from Uze et al., Cell Vol. 60, 225-234, 1990). The cDNA's weresubcloned into the pBlueBac 4.5/V5-His-TOPO vector (Invitrogen) and arecombinant Baculovirus obtained by homologous recombination, plaquepurification, and propagation in Sf9 cells. Sf9 cells were infected withthe recombinant Baculovirus and expression from the resulting cells wasobtained essentially as described in Example 2.

[0495] IFNAR1ec and IFNAR2ec protein was observed in culturesupernatants two to three days after infection of Sf9 cells withrecombinant baculovirus. The activity of soluble receptors was observedin an Interferon antagonist assay. Briefly, Hela cells containing theISRE element (as described in the Primary Assay above) are stimulatedwith a sub-maximal dose of human wild-type Interferon β in the presenceof varying concentrations of IFNARec supernatant. The antagonist effectof the supernatant is directly proportional to the amount of solublereceptor present.

[0496] IFNAR2ec was purified from filtered culture supernatants usingion exchange, and affinity chromatography. Culture supernatants positivefor IFNAR2ec were pH adjusted to 7.5 and loaded onto an anion exchangecolumn, and the bound recombinant protein was eluted using 500 mM NaCl.The partially pure IFNAR2ec was then diluted and pH adjusted to 8.0,before further purification using binding to a TALON™ Metal AffinityResin and elution with imidazol. The final preparation was frozen inaliquots. IFNAR1ec can be purified as described for IFNAR2ec with theexception that cation exchange chromatography at pH6.0 will be used asthe ion exchange step.

Example 11

[0497] Use of Soluble IFNAR2 for Purification and Pegylation ofInterferon-β and Variants Thereof

[0498] Purified IFNAR2 obtained as described in Example 9 is immobilizedeither through amino or carboxyl groups using e.g. CNBr-activatedSepharose 4B or EAH Sepharose 4B according to the manufacturer'sinstructions (Amersham Pharmacia Biotech, Affinity Chromatography,Principles and Methods, 18-1022-29, edition AB). It is criticallyimportant that the coupling method allows functional IFNAR2 to beimmobilized and this is tested through optimization of the couplingconditions (pH, coupling buffer, ratio of IFNAR 2 to activate matrixetc). Another critical parameter is the blocking of excess activegroups. Subsequently, testing of binding capacity by addition ofinterferon-β and measurement of breakthrough is carried out.

[0499] Optimally immobilized IFNAR2 is used for purification ofInterferon-β as follows. A 5 ml column with 1 mg IFNAR 2 immobilized perml gel is equilibrated with buffer A (20 mM phosphate, 300 mM NaCl, pH7). Then the column is loaded with a 2 mg Interferon-β sample in bufferA and subsequently washed with 5 column volumes buffer A. Elution isobtained by pumping 2 column volumes of buffer B onto the column.Fractions of 1 ml are collected and assayed for bioactivity. Optimalelution conditions are dependent on the immobilization method, butexamples of elution conditions include pH 1.5-3 (e.g. 0.1 M glycine pH2.3 in 0.5 M NaCl), pH 11.5-12, 3.5 M MgCl₂, 6M urea or the like.

Example 12

[0500] Use of Immobilized IFNAR2 for Pegylation of Interferon β(Variants)

[0501] In addition to the use described in Example 10, immobilized IFNAR2 may be used for optimal PEGylation, wherein PEGylation of the part ofInterferon-β or variants thereof interacting with the receptor isavoided.

[0502] A 5 ml column with 1 mg IFNAR 2 immobilized per ml gel isequilibrated with buffer A (20 mM phosphate, 300 mM NaCl, pH 7). Thenthe column is loaded with a 2 mg Interferon-β sample in buffer A andsubsequently washed with 5 column volumes buffer A. A solution ofactivated mPEG-SPA (1-50 mg/ml in buffer A) is pumped on the column andallowed to react for 15 min-12 h depending on temperature. One preferredrange of combination of residence time and temperature is 15-60 min,10-20 C, another is 30 min to 5 h, 2-8 C. After the indicated timeperiod, elution is obtained by pumping 2 column volumes of buffer B ontothe column. Fractions of 1 ml are collected and assayed for bioactivityusing the primary screening assay. Optimal elution conditions aredependent on the immobilization method, but examples of elutionconditions include pH 1.5-3 (e.g. 0.1 M glycine pH 2.3 in 0.5 M NaCl),pH 11.5-12, 3.5 M MgCl₂, 6M Urea or the like.

Example 13

[0503] Antiviral Activity of Pegylated Variant

[0504] The pegylated IFN-β variant protein, K19R+K45R+K123R, was assayedusing the antiviral bioassay. Wild-type and variant proteins were addedto A549 cells in concentrations from 10-0.0001 IU/mL in triplicatecultures.

[0505] The pegylated IFN-β variant showed total inhibition of EMC virusinduced cell death at a concentration of 3 IU/mL, with an EC50 of 0.13IU/mL (FIG. 1). The wild-type standard shows virus inhibition with anEC50 of 1.4 IU/mL.

[0506] These results demonstrate that the pegylation of the modifiedinterferon β polypeptide resulted in a conjugate with full anti-viralactivity.

Example 14

[0507] Antibody Neutralisation of Glycosylated Variant

[0508] The antibody neutralisation of wild-type and glycosylated IFN-βvariant protein, Q49N+Q51T+F111N+R113T, was assayed using the ISREneutralisation assay. Interferon β wild-type and variant proteins (infive fold dilutions starting at 12500 IU/mL) were incubated withpolyclonal rabbit anti-interferon β antibody (PBL BiomedicalLaboratories) in concentrations 0, 40 and 200 ng/mL.

[0509] In the presence of 200 ng/mL polyclonal rabbit anti-serum theactivity of the wild type interferon β protein was reduced 11.8 timeswhereas the activity of the glycosylated interferon β variant only wasreduced 3.0 times. Thus the degree of antibody recognition of theinterferon β variant was reduced by 75% of the wt level, see Table 1below. These results demonstrate that the recognition of theglycosylated mutant interferon β by polyclonal antibodies raised inanimals immunised with wild-type human interferon 13 is highly reduced.Thus, a large portion of the immunogenic epitopes in wild-type humaninterferon β have been removed/shielded by the modifications made in thevariant molecule. TABLE 1 Reduction of Antibody conc. antibody (ng/mL)Protein EC50 Fold inhibition neutralisation 0 wt 0.00039 — — variant0.00020 — — 40 wt 0.00190 4.8 — variant 0.00020 1.0 79% 200 wt 0.0046111.8 — variant 0.00059 3.0 75%

Example 15

[0510] Construction and Expression of Interferon β Molecules withModifled N-Terminal

[0511] N-terminally modified variants of interferon β were constructedas described in the preceding examples.

[0512] For the construction of an expression plasmid for the interferonβ variant, INFB S(−1)A+M1Q the following primers were used: (SEQ ID NO37) CBProFpr110: AAC TGG ATC CAG CCA CCA TGA CCA ACA AGT GCC TGC TCC AGATCG CCC TGC TCC TGT GCT TCA GCA CCA CGG CCC TAG CCC AGA GCT AC, and (SEQID NO 26) CBProFpr42,

[0513] For the construction of an expression plasmid for the interferonvariant, INFβ S(−1)AQ (indicating substitution of the S residue locatedin position (−1) with an A and a Q residue) the following primers wereused: (SEQ ID NO 38) CBProFpr109: AAC TGG ATC CAG CCA CCA TGA CCA ACAAGT GCC TGC TCC AGA TCG CCC TGC TCC TGT GCT TCA GCA CCA CGG CCC TAG CCCAGA TGA GCT AC and (SEQ ID NO 26) CBProFpr42.

[0514] To test the activity of these variants the respective plasmids;pF154 and pF163 were transfected into CHO K1 cells using Lipofectamine2000 (Life Technologies, USA) as transfection reagent. The supernatantswere harvested 24 hours post transfection and assayed in the primaryactivity assay and in the ELISA as described in the Materials andMethods section.

[0515] The following results were obtained: INFB S-1A + M1Q (pF154):Activity: 106410 IU/ml ELISA:   333 ng/ml Specific activity:   3.2 × 10⁸IU/mg INFB S-1AQ (pF163): Activity: 90634 UI/ml ELISA:  193 ng/mlSpecific activity:   4.7 × 10⁸ IU/mg

[0516] These molecules are as active as wild type human interferon β.

Example 16

[0517] Preparation of Pegylated IFN-β Variants

[0518] 50 microliters of a 0.3 mg/ml solution of recombinant human IFN-βpolypeptide comprising the mutations Q49N+Q51T+K19R+K45R+K123R in 50 mMNa-acetate, 35% ethylene glycol, pH 5.5 were mixed with 10 μl 0.5 MNa-phosphate, pH 8.0 and 20 μl 50 mM Na-phosphate, 0.1 M NaCl, 30%ethylene glycol, pH 8.0 containing 0.02 mg/ml SPA-mPEG (N-succinimidylPropionate methoxy polyethylene glycol). This corresponds to a 10 molarexcess of SPA-mPEG to IFN-β.

[0519] After ½ hour with gentle rotation at room temperature, thereaction was quenched by addition of 5 μl 20 mM Glycine, pH 8.0. At thisstage, the reaction mixture contained a mixture of unmodified as well aspegylated forms of recombinant human IFN-β.

[0520] Activity:

[0521] In vitro testing using the primary screening assay demonstratedthat the pegylated material retained 40% activity, as compared to theunmodified recombinant human IFN-β.

[0522] In another experiment, 50 μl of a 0.14 mg/ml solution ofrecombinant human IFN β polypeptide comprising the mutations Q49N+Q51Tin 50 mM Na-acetate, 35% ethylene glycol, pH 5.5 was mixed with 10 μl0.5 M Na-phosphate, pH 8.0 and 20 μl 50 mM Na-phosphate, 0.1 M NaCl, 30%ethylene glycol, pH 8.0 containing 0.03 mg/ml SPA-mPEG. This gave a 10molar excess of SPA-MPEG to IFN-β.

[0523] After ½ hour with gentle rotation at room temperature, thereaction was quenched by addition of 5 μl 20 mM Glycine, pH 8.0. At thisstage, the reaction mixture contained a mixture of unmodified as well aspegylated forms of recombinant human IFN-β.

[0524] Activity:

[0525] In vitro testing using the primary screening assay demonstratedthat the pegylated material retained 20% activity, as compared to theunmodified recombinant human IFN-β.

[0526] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be clear to oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention. For example, all the techniques, methods,compositions, apparatus and systems described above may be used invarious combinations. All publications, patents, patent applications, orother documents cited in this application are incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication, patent, patent application, or other documentwere individually indicated to be incorporated by reference for allpurposes.

1 45 1 840 DNA Homo sapiens 1 acattctaac tgcaaccttt cgaagcctttgctctggcac aacaggtagt aggcgacact 60 gttcgtgttg tcaacatgac caacaagtgtctcctccaaa ttgctctcct gttgtgcttc 120 tccactacag ctctttccat gagctacaacttgcttggat tcctacaaag aagcagcaat 180 tttcagtgtc agaagctcct gtggcaattgaatgggaggc ttgaatactg cctcaaggac 240 aggatgaact ttgacatccc tgaggagattaagcagctgc agcagttcca gaaggaggac 300 gccgcattga ccatctatga gatgctccagaacatctttg ctattttcag acaagattca 360 tctagcactg gctggaatga gactattgttgagaacctcc tggctaatgt ctatcatcag 420 ataaaccatc tgaagacagt cctggaagaaaaactggaga aagaagattt caccagggga 480 aaactcatga gcagtctgca cctgaaaagatattatggga ggattctgca ttacctgaag 540 gccaaggagt acagtcactg tgcctggaccatagtcagag tggaaatcct aaggaacttt 600 tacttcatta acagacttac aggttacctccgaaactgaa gatctcctag cctgtgcctc 660 tgggactgga caattgcttc aagcattcttcaaccagcag atgctgttta agtgactgat 720 ggctaatgta ctgcatatga aaggacactagaagattttg aaatttttat taaattatga 780 gttattttta tttatttaaa ttttattttggaaaataaat tatttttggt gcaaaagtca 840 2 166 PRT Homo sapiens 2 Met SerTyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 CysGln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 LysAsp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 GlnPhe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 AsnIle Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105110 Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr130 135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn ArgLeu 145 150 155 160 Thr Gly Tyr Leu Arg Asn 165 3 70 DNA ArtificialSequence Description of Artificial Sequence primer 3 ggctagcgtttaaacttaag cttcgccacc atgaccaaca agtgcctgct ccagatcgcc 60 ctgctcctgt 704 70 DNA Artificial Sequence Description of Artificial Sequence primer 4acaacctgct cggcttcctg cagaggagtt cgaacttcca gtgccagaag ctcctgtggc 60agctgaacgg 70 5 70 DNA Artificial Sequence Description of ArtificialSequence primer 5 gaacttcgac atccccgagg aaatcaagca gctgcagcag ttccagaaggaggacgccgc 60 tctgaccatc 70 6 70 DNA Artificial Sequence Description ofArtificial Sequence primer 6 ttccgccagg actccagctc caccggttgg aacgagaccatcgtggagaa cctgctggcc 60 aacgtgtacc 70 7 70 DNA Artificial SequenceDescription of Artificial Sequence primer 7 aggagaagct ggagaaggaggacttcaccc gcggcaagct gatgagctcc ctgcacctga 60 agcgctacta 70 8 70 DNAArtificial Sequence Description of Artificial Sequence primer 8ggagtacagc cactgcgcct ggaccatcgt acgcgtggag atcctgcgca acttctactt 60catcaaccgc 70 9 70 DNA Artificial Sequence Description of ArtificialSequence primer 9 caccacactg gactagtgga tccttatcag ttgcgcaggt agccggtcaggcggttgatg 60 aagtagaagt 70 10 70 DNA Artificial Sequence Description ofArtificial Sequence primer 10 aggcgcagtg gctgtactcc ttggccttcaggtagtgcag gatgcggcca tagtagcgct 60 tcaggtgcag 70 11 70 DNA ArtificialSequence Description of Artificial Sequence primer 11 ctccttctccagcttctcct ccagcacggt cttcaggtgg ttgatctggt ggtacacgtt 60 ggccagcagg 7012 70 DNA Artificial Sequence Description of Artificial Sequence primer12 gagctggagt cctggcggaa gatggcgaag atgttctgca gcatctcgta gatggtcaga 60gcggcgtcct 70 13 70 DNA Artificial Sequence Description of ArtificialSequence primer 13 cctcggggat gtcgaagttc atcctgtcct tcaggcagtactccaggcgc ccgttcagct 60 gccacaggag 70 14 70 DNA Artificial SequenceDescription of Artificial Sequence primer 14 caggaagccg agcaggttgtagctcatcga tagggccgtg gtgctgaagc acaggagcag 60 ggcgatctgg 70 15 70 DNAArtificial Sequence Description of Artificial Sequence primer 15ctgctccaga tcgccctgct cctgtgcttc agcaccacgg ccctatcgat gaagcaccag 60caccagcatc 70 16 70 DNA Artificial Sequence Description of ArtificialSequence primer 16 cactgcttac tggcttatcg aaattaatac gactcactatagggagaccc aagctggcta 60 gcgtttaaac 70 17 70 DNA Artificial SequenceDescription of Artificial Sequence primer 17 caggaagccg agcaggttgtagctcatctg ttggtgttga tgttggtgct gatgctggtg 60 ctggtgcttc 70 18 70 DNAArtificial Sequence Description of Artificial Sequence primer 18agcagggcga tctggagcag gcacttgttg gtcatggtgg cgaagcttaa gtttaaacgc 60tagccagctt 70 19 40 DNA Artificial Sequence Description of ArtificialSequence primer 19 ccgtcagatc ctaggctagc ttattgcggt agtttatcac 40 20 32DNA Artificial Sequence Description of Artificial Sequence primer 20gagctcggta ccaagctttt aagagctgta at 32 21 77 DNA Artificial SequenceDescription of Artificial Sequence primer 21 gctgaacggg cgcctggagtactgcctgaa ggacaggatg aacttcgaca tccccgagga 60 aatccgccag ctgcagc 77 2235 DNA Artificial Sequence Description of Artificial Sequence primer 22tctccacgcg tacgatggtc caggcgcagt ggctg 35 23 70 DNA Artificial SequenceDescription of Artificial Sequence primer 23 caccacactg gactagtggatccttatcag ttgcgcaggt agccggtcag gcggttgatg 60 aagtagaagt 70 24 31 DNAArtificial Sequence Description of Artificial Sequence primer 24catcagcttg ccggtggtgt tgtcctcctt c 31 25 31 DNA Artificial SequenceDescription of Artificial Sequence primer 25 gaaggaggac aacaccaccggcaagctgat g 31 26 40 DNA Artificial Sequence Description of ArtificialSequence primer 26 cacactggac tagtaagctt ttatcagttg cgcaggtagc 40 27 47DNA Artificial Sequence Description of Artificial Sequence primer 27gaggagttcg aacttccagt gccagcgcct cctgtggcag ctgaacg 47 28 29 DNAArtificial Sequence Description of Artificial Sequence primer 28cgcggatcca tatgaccaac aagtgcctg 29 29 24 DNA Artificial SequenceDescription of Artificial Sequence primer 29 cgcggatcct tatcagttgc gcag24 30 33 DNA Artificial Sequence Description of Artificial Sequenceprimer 30 tttaaactgg atccagccac catgaccaac aag 33 31 63 DNA ArtificialSequence Description of Artificial Sequence primer 31 cggccatagtagcgcttcag gtgcagggag ctcatcagct tgccggtggt gttgtcctcc 60 ttc 63 32 63DNA Artificial Sequence Description of Artificial Sequence primer 32gaaggaggac aacaccaccg gcaagctgat gagctccctg cacctgaagc gctactatgg 60 ccg63 33 30 DNA Artificial Sequence Description of Artificial Sequenceprimer 33 ggcgtcctcc ttggtgaagt tctgcagctg 30 34 39 DNA ArtificialSequence Description of Artificial Sequence primer 34 atatatcccaagcttttatc agttgcgcag gtagccggt 39 35 30 DNA Artificial SequenceDescription of Artificial Sequence primer 35 cagctgcaga acttcaccaaggaggacgcc 30 36 34 DNA Artificial Sequence Description of ArtificialSequence primer 36 cgcggatcca gccaccatga ccaacaagtg cctg 34 37 89 DNAArtificial Sequence Description of Artificial Sequence primer 37aactggatcc agccaccatg accaacaagt gcctgctcca gatcgccctg ctcctgtgct 60tcagcaccac ggccctagcc cagagctac 89 38 92 DNA Artificial SequenceDescription of Artificial Sequence primer 38 aactggatcc agccaccatgaccaacaagt gcctgctcca gatcgccctg ctcctgtgct 60 tcagcaccac ggccctagcccagatgagct ac 92 39 6 PRT Artificial Sequence Description of ArtificialSequence peptide tag 39 His His His His His His 1 5 40 8 PRT ArtificialSequence Description of Artificial Sequence peptide tag 40 Met Lys HisHis His His His His 1 5 41 10 PRT Artificial Sequence Description ofArtificial Sequence peptide tag 41 Met Lys His His Ala His His Gln HisHis 1 5 10 42 14 PRT Artificial Sequence Description of ArtificialSequence peptide tag 42 Met Lys His Gln His Gln His Gln His Gln His GlnHis Gln 1 5 10 43 10 PRT Artificial Sequence Description of ArtificialSequence peptide tag 43 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 1044 8 PRT Artificial Sequence Description of Artificial Sequence peptidetag 44 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 45 9 PRT Artificial SequenceDescription of Artificial Sequence peptide tag 45 Tyr Pro Tyr Asp ValPro Asp Tyr Ala 1 5

What is claimed is:
 1. A conjugate exhibiting interferon β activity,which conjugate comprises at least one first non-polypeptide moietycovalently attached to an interferon β polypeptide variant, whichinterferon β polypeptide variant differs from a wild-type humaninterferon β by at least one introduction and at least one removal of anamino acid residue, which amino acid residue comprises an attachmentgroup for the first non-polypeptide moiety.
 2. The conjugate of claim 1,wherein the first non-polypeptide moiety is selected from the groupconsisting of: a polymer molecule, a lipophilic compound, a sugar moietyand an organic derivatizing agent.
 3. The conjugate of claim 2, whereinthe polymer molecule comprises linear or branched polyethylene glycol.4. The conjugate of claim 1, wherein the first non-polypeptide moietycomprises a polymer molecule having an attachment group selected fromthe group consisting of: lysine, aspartic acid, glutamic acid andcysteine.
 5. The conjugate of claim 4, wherein the first non-polypeptidemoiety is a polymer molecule having lysine as an attachment group.
 6. Aconjugate exhibiting interferon β activity, which conjugate comprises atleast one first non-polypeptide moiety conjugated to at least one lysineresidue of an interferon β polypeptide variant, which interferon βpolypeptide variant differs from a wild-type human interferon β by atleast one introduction or removal of a lysine residue.
 7. The conjugateof claim 6, which interferon β polypeptide variant differs from awild-type human interferon β by introduction and removal of a lysineresidue.
 8. The conjugate of claim 6, wherein the lysine residue removedis selected from one or more residue selected from the group consistingof: K19, K33, K45, K52, K99, K105, K108, K115, K123, K134 and K136. 9.The conjugate of claim 8, wherein the lysine residue has beensubstituted with an arginine or a glutamine residue.
 10. The conjugateof claim 6, wherein the interferon β polypeptide variant comprises oneof the following sets of mutations: K19R+K45R+K123R; K19Q+K45R+K123R;K19R+K45Q+K123R; K19R+K45R+K123Q; K19Q+K45Q+K123R; K19R+K45Q+K123Q;K19Q+K45R+K123Q; K19Q+K45Q+K123Q; K45R+K123R; K45Q+K123R; K45Q+K123Q;K45R+K123Q; K19R+K123R; K19Q+K123R; K19R+K123Q; K19Q+K123Q; K19R+K45R;K19Q+K45R; K19R+K45Q; K19Q+K45Q; K52R+K134R; K99R+K136R;K33R+K105R+K136R; K52R+K108R+K134R; K99R+K115R+K136R;K19R+K33R+K45R+K123R; K19R+K45R+K52R+K123R; K19R+K33R+K45R+K52R+K123R;or K19R+K45R+K52R+K99R+K123R.
 11. The conjugate of claim 6, wherein alysine residue has been introduced in a position selected from the groupconsisting of: N4, F8, L9, R11, S12, F15, Q16, Q18, L20, W22, Q23, G26,R27, L28, E29, Y30, L32, R35, M36, N37, D39, P41, E42, E43, L47, Q48,Q49, T58, Q64, N65, F67, A68, R71, Q72, D73, S75, S76, G78, N80, E81,I83, E85, N86, A89, N90, Y92, H93, H97, T100, L102, E103, L106, E107,E109, D110, F111, R113, G114, L116, M117, L120, H121, R124, G127, R128,L130, H131, E137, Y138, H140, I145, R147, V148, E149, R152, Y155, F156,N158, R159, G162, Y163, R165 and N166 of SEQ ID NO
 2. 12. The conjugateof claim 11, wherein the interferon β polypeptide variant comprises atleast one substitution selected from the group consisting of: N4K, R11K,G26K, R27K, Q48K, Q49K, R71K, D73K, S75K, E85K, A89K, Y92K, H93K, F111K,R113K, R113K, L116K, R124K, G127K and Y155K.
 13. The conjugate of claim12, wherein the substitution is selected from the group consisting of:Q49K and F111K.
 14. The conjugate of claim 6, comprising at least twointroduced lysine residues.
 15. The conjugate of claim 11, wherein theinterferon β polypeptide variant further comprises the removal of atleast one lysine residue.
 16. The conjugate of claim 15, wherein the atleast one lysine residue is selected from the group consisting of: K19,K33, K45, K52, K99, K105, K108, K115, K123, K134 and K136.
 17. Theconjugate of claim 15, comprising one of the following sets ofmutations: K19R+K45R+F111K+K123R; K19R+K45R+Q49K+F111K+K123R;K19R+K45R+Q49K+K123R; K19R+K45R+F111K; K19R+K45R+Q49K+F111K;K19R+Q49K+K123R; K19R+Q49K+F111K+K123R; K45Q+F111K+K123Q;K45R+Q49K+K123R; or K45R+Q49K+F111K+K123R.
 18. The conjugate of claim 1or 6, wherein the non-polypeptide moiety comprises a polymer selectedfrom the group consisting of: SS-PEG, NPC-PEG, aldehyd-PEG, mPEG-SPA,PEG-SCM and mPEG-BTC.
 19. A conjugate exhibiting interferon β activity,which conjugate comprises at least one first non-polypeptide moietyconjugated to at least one cysteine residue of an interferon βpolypeptide variant, which interferon β polypeptide variant differs froma wild type interferon β polypeptide by the introduction of at least onecysteine residue at a position selected from the group consisting of:F8, L9, R11, S12, F15, Q16, Q18, L20, W22, L28, L32, M36, P41, T58, Q64,N65, F67, I83, E85, N86, A89, N90, Y92, H93, H97, T100, L102, E103,L106, M117, L120, H121, R124, G127, R128, L130, H131, H140, I145, R147,V148, E149, R152, Y155, and F156 of SEQ ID NO
 2. 20. The conjugate ofclaim 19, wherein the interferon β polypeptide variant further comprisesremoval of at least one cysteine residue.
 21. The conjugate of claim 20,wherein the cysteine residue removed comprises C17.
 22. The conjugate ofclaim 19, wherein the interferon β polypeptide variant comprises atleast one of the mutations C17S or N80C.
 23. The conjugate of claim 19,wherein the interferon β polypeptide variant comprises the mutationsC17S and N80C.
 24. The conjugate of claim 19, wherein the firstnon-polypeptide moiety is a polymer molecule.
 25. A conjugate exhibitinginterferon β activity, which conjugate comprises at least one firstnon-polypeptide moiety having an acid group as an attachment group,which moiety is conjugated to at least one aspartic acid or glutamicacid residue of an interferon β polypeptide variant, which interferon βpolypeptide variant differs from a wild-type human interferon β by atleast one introduction or removal of an aspartic acid or a glutamic acidresidue.
 26. The conjugate of claim 25, wherein at least one amino acidresidue selected from the group consisting of: N4, L5, L6, F8, L9, Q10,R11, S12, S13, F15, Q16, Q18, K19, L20, W22, Q23, L24, N25, G26, R27,Y30, M36, Q46, Q48, Q49, I66, F67, A68, I69, F70, R71, S75, T82, I83,L87, A89, N90, V91, Y92, H93, Q94, I95, N96, H97, K108, F111, L116,L120, K123, R124, Y126, G127, R128, L130, H131, Y132, K134, A135, H140,T144, R147, Y155, F156, N158, R159, G162, Y163 and R165 of SEQ ID NO 2is substituted with an aspartic acid residue or a glutamic acid residue.27. The conjugate of claim 25, comprising introduction of at least twointroduced aspartic acid or glutamic acid residues.
 28. The conjugate ofclaim 25, comprising at least two first non-polypeptide moieties. 29.The conjugate of claim 25, wherein the first non-polypeptide moiety is apolymer molecule.
 30. The conjugate of claim 29, wherein the secondnon-polypeptide moiety comprises a sugar moiety.
 31. The conjugate ofclaim 34, wherein the amino acid sequence of the interferon βpolypeptide variant further comprises at least one of an introduction ora removal of an in vivo glycosylation site.
 32. The conjugate of claim31, wherein the first non-polypeptide moiety is a polymer moleculehaving lysine as an attachment group.
 33. The conjugate of claim 1, 6,19 or 25, which conjugate comprises a second non-polypeptide moiety. 34.The conjugate of claim 33, wherein the polypeptide comprises at leastone removed amino acid residue comprising an attachment group for thefirst non-polypeptide moiety, and at least one introduced amino acidresidue comprising an attachment group for the second non-polypeptidemoiety.
 35. The conjugate of claim 33, wherein the amino acid sequenceof the interferon β polypeptide variant comprises at least two removedamino acid residues comprising attachment groups for the firstnon-polypeptide moiety and at least one introduced amino acid residuecomprising an attachment group for the second non-polypeptide moiety.36. The conjugate of claim 33, wherein the second non-polypeptide moietycomprises a sugar moiety.
 37. A conjugate exhibiting interferon βactivity, which conjugate comprises at least one polymer molecule and atleast one sugar moiety covalently attached to an interferon βpolypeptide variant, which interferon β polypeptide variant differs froma wild-type human interferon β by (a) at least one introduction orremoval of an amino acid residue, which amino acid residue comprises anattachment group for the polymer molecule, and (b) at least oneintroduction or removal of an amino acid residue, which amino acidresidue comprises an attachment group for the sugar moiety, with theproviso that when the attachment group for the polymer molecule is acysteine residue, and the sugar moiety is an N-linked sugar moiety,introduction of a cysteine residue does not destroy an N-glycosylationsite.
 38. The conjugate of claim 37, wherein the polymer molecule haslysine as an attachment group.
 39. The conjugate of claim 38, whereinthe polypeptide variant comprises at least one removed amino acidresidue comprising an attachment group for the first non-polypeptidemoiety and at least one introduced amino acid residue comprising anattachment group for the second non-polypeptide moiety.
 40. Theconjugate of claim 37, wherein the interferon β polypeptide variantcomprises one of the following sets of mutations:K19R+K45R+Q49N+Q51T+F111N+R113T+K123R; K19R+K45R+Q49N+Q51T+F111N+R113T;or K19R+K45R+Q49N+Q51T+K123R.
 41. The conjugate of claim 1, 6, 19, 25 or37, wherein the interferon β polypeptide variant comprises a modifiedN-terminus, which modified N-terminus is unavailable for conjugation toa non-polypeptide moiety.
 42. A conjugate exhibiting interferon βactivity, which conjugate comprises an interferon β polypeptide variant,which interferon β polypeptide variant differs from a wild-type humaninterferon β by introduction of at least one glycosylation site, theconjugate further comprising at least one un-PEGylated sugar moietyattached to the introduced glycosylation site.
 43. A conjugateexhibiting interferon β activity, which conjugate comprises aninterferon β polypeptide variant, which interferon β polypeptide variantdiffers from a wild-type human interferon β by at least one introductionor removal of a glycosylation site by introducing or removing one ormore amino acid residues comprising a glycosylation site in a positionthat is occupied by an amino acid residue having at least 25% of itsside chain surface exposed in wildtype human interferon β.
 44. Theconjugate of claim 42 or 43, wherein the interferon β polypeptidevariant comprises at least one mutation selected from the groupconsisting of: S2N+N4T, L9N+R11T, R11N, S12N+N14T, F15N+C16S, Q16N+Q18T,K19N+L21T, Q23N+H25T, G26N+L28T, R27N+E29T, L28N+Y30T, D39T, K45N+L47T,Q46N+Q48T, Q48N+F50T, Q49N+Q51T, Q51N+E53T, R71N+D73T, Q72N, D73N, S75N,S76N+G78T, L88T, Y92T, N93N+195T, L98T, E103N+K105T, E104N+L106T,E107N+E109T, K108N+D110T, D110N, F111N+R113T and L116N.
 45. Theconjugate of claim 44, wherein the interferon β polypeptide comprisesone of the following sets of substitutions: Q49N+Q51T;Q49N+Q51T+F111N+R113T; or Q49N+Q51T+R71N+D73T+F111N+R113T.
 46. Theconjugate of claim 42 or 43, wherein the amino acid sequence furtherdiffers by removal of an amino acid comprising a glycosylation site. 47.The conjugate of claim 46, wherein the glycosylation site comprises anN-glycosylation site.
 48. A conjugate exhibiting interferon β activity,which conjugate comprises a sugar moiety covalently attached to aninterferon β polypeptide variant, which interferon β polypeptide variantdiffers from a wild-type human interferon β by removal of at least oneglycosylation site.
 49. The conjugate of claim 43 or 48, wherein anN-glycosylation site is removed by the mutation N80C.
 50. The conjugateof claim 1, 6, 19, 25, 37 or 43, wherein the interferon β polypeptidefurther comprises at least one substitution in a position selected fromamong M1, C17, N80 or V101.
 51. The conjugate of claim 50, wherein theinterferon β polypeptide variant comprises at least one substitutionselected from M1del, M1K or C17S.
 52. A nucleotide sequence encoding aninterferon β polypeptide variant, which interferon β polypeptide variantcomprises component of a conjugate exhibiting interferon β activity. 53.A host cell comprising the nucleotide sequence of claim
 52. 54. Anexpression vector comprising the nucleotide sequence of claim
 52. 55. Ahost cell comprising the expression vector of claim
 54. 56. The hostcell of claim 53 or 55, which host cell comprises a CHO cell, a BHKcell, a HEK293 cell or an SF9 cell.
 57. A method of reducingimmunogenicity or increasing functional in vivo half-life or serumhalf-life of an interferon β polypeptide variant, the method comprising:(a) introducing an amino acid residue comprising an attachment group fora first non-polypeptide moiety into a position exposed at the surface ofan interferon β polypeptide that does not contain such group; or (b)removing an amino acid residue comprising an attachment group for thefirst non-polypeptide moiety; and conjugating the interferon βpolypeptide variant with the first non-polypeptide moiety.
 58. Themethod of claim 57, wherein the non-polypeptide moiety is selected fromthe group consisting of a polymer molecule, a sugar moiety, a lipophilicgroup and an organic derivatizing agent.
 59. A method for preparing aconjugate exhibiting interferon β activity, the method comprising:contacting an interferon β polypeptide variant with a firstnon-polypeptide moiety under conditions conducive for conjugation; andrecovering the conjugate exhibiting interferon β activity.
 60. Apharmaceutical composition comprising a conjugate exhibiting interferonβ activity and a pharmaceutically acceptable diluent, carrier, excipientor adjuvant.
 61. The pharmaceutical composition of claim 60, wherein thepharmaceutical composition is effective for the treatment of one or morediseases.
 62. The pharmaceutical composition of claim 61, wherein thepharmaceutical composition is effective for the treatment of multiplesclerosis.
 63. A method for treating a mammal with multiple sclerosiscomprising administering an effective amount of the pharmaceuticalcomposition of claim
 60. 64. A method for treating a mammal havingcirculating antibodies against interferon β 1a and/or 1b,which methodcomprises administering an effective amount of the pharmaceuticalcomposition of claim
 60. 65. The method of claim 63 or 64, wherein themammal is a human.
 66. A cell culture comprising a) a host celltransformed with a nucleotide sequence encoding an interferon βpolypeptide variant; and, b) a culture medium.
 67. The cell culture ofclaim 66, wherein the culture medium comprises an interferon βpolypeptide variant produced by expressing the nucleotide sequence. 68.The cell culture of claim 67, wherein the concentration of theinterferon β polypeptide variant is at least 800,000 IU/ml of medium.69. The cell culture of claim 67, wherein the concentration of theinterferon β polypeptide variant is in the range of 800,000-3,500,000IU/ml medium.
 70. A cell culture comprising a host cell of claim 53 or55.
 71. A method of producing an interferon β polypeptide variant, themethod comprising: (a) culturing a cell expressing an interferon βpolypeptide variant in a culture medium, such that the concentration ofthe interferon β polypeptide variant in the medium is at least 800,000IU/ml medium; and (b) recovering the interferon β polypeptide variant.72. The method of claim 71, wherein the concentration of the interferonβ polypeptide variant is between 800,000 and 3,000,000 IU/ml medium.